TECHNICAL FIELD This invention relates to a composition comprising phytohaemagglutinin, and glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof.

Also contemplated is a pharmaceutical composition, use thereof and methods for obtaining mature gastrointestinal tracts in premature or neonatal mammals and for treating uremic, predialysis, dialysis's or transplantation patients with renal failure.

BACKGROUND OF THE INVENTION The digesetive system of neonatal mammals The gastrointestinal tract, GIT, is the tract pertaining to or communicating with the stomach and intestine. The GIT and its accessory glands undergo major development during the neonatal period in mammals.

The digestive system of neonatal pigs In piglets, the neonatal development is illustrated by a shift in expression of enterocytes brush border disaccaridases with decreased lactase and increased surcease and maltase activities. Moreover, enterocytes with a high endocytic activity, the foetal-type, are gradually replaced during the first 3 weeks of life by new adult-type cells with marked reduced endocytosis (Klein, 1989). These maturational changes gradually progress with age and become evident at weaning, when the gut has to change its digestive and absorptive abilities to effectively utilise the weaning diet instead of the more easily digestible milk. Extensive structural changes are seen in the intestine at this time, with a decreased villi height and increased crypt dept. In addition, the stomach acid secretion amplifies and the amount of gut enzymes increases, reflecting an elevated pancreatic function (Henning et al., I994).

Effects of early weaning in a mammal Due to the early abrupt weaning that is generally practised in today's pig production, a natural development of the gut is not allowed. In today's pig production the gut function is unmature at early weaning and does not match the requirements of digestion and absorption of the weaning food. Inefficient (incomplete) digestion will cause overgrowth of gut bacteria because of high availability of substrate for the gut bacteria.

Therefore, the weaning period is hazardous for young pigs, which is manifested by the high frequency of the gastrointestinal tract (GIT) disorders, resulting in reduced

weight gain and poorer food utilisation at weaning. Moreover, the GIT disorders are one of the most important causes of animal death loss in young pigs.

Postweaning diarrhoea in pigs is a serious and complex problem from the standpoint of health of the pigs and of economics concerning this process. The traditional strategy to treat or minimise the weaning problems are generally based on antibiotic treatment or feed supplementation with antibiotics after weaning, the feeding of easy digest weaning food or creep feeding before weaning to create an adaptation of GIT. Though, for early weaning, when pigs are not able to consume enough creep feed before being weaned, this is not working.

Thus, weaning is a period of transition, hazardous to an animal's health in relation to the gut disturbances that occur due to lack of luminal stimulation following inadequate feed intake and due to immature gastrointestinal structure and function.

In the EC yearly, about 200 millions pigs need to go through weaning process and about 10-20% of them is dying because of weaning diarrhoea. Economical losses are can be estimated to about 2000 millions EURO.

Renal failure and dialysis The kidneys filter the blood e. g., from phosphate and metabolic waste products such as urea-containing nitrogen-and further excrete it as urine. Other waste products may be creatinin. Renal failure leads to several problems that generally are treated with by 1) protein free diet and electrolyte supplementation in early stages of uremia development when kidney still is partially in function, or, 2) dialysis or by kidney transplantation in end stage of uremia when kidney function is severely abolished. One indication that needs dialysis, is an increased level of nitrogen (N) in the form of urea, as well as an increase of other metabolic waste products.

Renal failure or renal malfunction is the state when kidneys fail to clean the blood from waste products. The kidney failure causes an accumulation of the toxic waste products in the blood. The kidneys normally have excess cleaning capacity and the renal capacity could be 50% of normal before symptoms occur. Symptoms are itching, tiredness, nausea, vomiting, loss of appetite leading to malnutrition.

Renal failure is often associated with diabetes and high blood pressure.

The dialysis procedure will reduce the pressure from the waste products on the kidneys. Still, it is a time consuming procedure, which the patient may need to perform several times a week. The patient undergoing a dialysis procedure needs medical attention and the procedure is both costly and time consuming.

There are two forms of renal failure, acute and chronic renal failure (ACF and CRF). Acute renal failure can normally be reversed, while chronic normally

progresses. CRF is divided in pre-dialysis and active treatment of uremia using e. g. dialysis or transplantation. There exists no exact definition of pre-dialysis as to the starting point but normally pre-dialysis is defined as the period in time between that renal failure have been diagnosed and when active treatment is initiated. Dialysis and transplantation is considered as active treatment.

Treatments in the early stage, if diagnosed, are protein restriction diet along with recommendation of reduced natrium, potassium, phosphate and water intake.

The objectives of such an early treatment are to delay the onset of active treatment of uremia through a delay of the progression of renal failure and reducing the uremic symptoms. The primary diagnosis of renal disease is subject to treatment.

Normally renal failure progress and at a certain stage, based on the individual's clinical situation, active treatment is initiated. Active treatment is e. g. hemo dialysis (HD), Continuous Ambulatory Peritoneal Dialysis (CAPD) or transplantation.

It is estimated that 1. 375 000 patients globally have reached ESRD. Out of these, 940 000 is undergoing HD treatment, 120 000 PD and 315 000 are living with donor kidneys.

The US accounted for 272 000 dialysis patients, Japan 209 000, Germany 52 000, Brazil 48 000 and Italy 41 000. The ESRD population grows by approximately 8% per year.

In the US alone the estimated number of pre-dialysis patients is 708 000 in 1990. The number has been growing significantly since, and some estimates point at 5-7 million patients world-wide, where about 1. 5-2 million of these are diagnosed.

The exact numbers depend on the definition used. Related to this, some 30 million people are at risk of developing renal failure.

Lectins Lectins, or hemagglutinins, are proteins obtained from plant and animal sources, particularly from the seeds of leguminous plants. The lectins have binding sites for specific mono-or oligosaccharides.

Lectins, such as concanavalin A and wheat germ agglutinin, are widely used as analytical and preparative agents in the study of glycoproteins. The red kidney bean (Phaseolus vulgaris) is the source of the lectin phytohaemagglutinin, PHA.

PHA is unique in recognizing large, complex carbohydrate structures (Kaneda et al., JBC 277 : 16928-16935, 2002), compared to most other lectins that mostly recognize descrete sugar linkages.

Plant lectins resist degradation by digestive enzymes and bacteria and survive passage through the alimentary tract. Since the mammalian gut surface is highly glycosylated, ingested lectins interact with different parts of the digestive system and exert powerful effects on its structure, function, digestive capacity, immune

status, bacterial flora and hormonal status. Most lectins are powerful gut growth factors and induce hyperplastic reversible growth, and faster epithelial cell turnover (Bardooz S. et el., (1995) Gut 37 : 353-360 ; Herzig IL. H. et al., (1997), Gut 41 : 333- 338.). Lectins may bind to e. g. Goblet cells and stimulate their secretion of mucins- this will contribute to the overall nutrient cost. If this increased demand for nutrients cannot be met by the diet, the body reserves will be mobilised. The depression of the overall nutritional performance at high dietary lectin concentrations is similar to that caused by nutritional toxins (anti-nutritional factors, ANF).

PHA has been shown to bind to carbohydrate structures on the gut epithelium in adult rats and thereby potently promote the growth and maturation of the GIT and the pancreas. It is also known that the red kidney bean lectin has similar growth- promoting effects accelerating the structural and functional maturation of the gut in pigs well before weaning (Rådberg et al., J. Anim. Sci., 2001, 79 : 2669-2678).

Enterocytes A proper and effective function of the gastro-intestinal tract (GIT) is provided by epithelium of the small intestine. The elementary components of epithelium in e. g. the small bowel are enterocytes. Enterocytes are cells of the intestinal epithelium. The enterocytes are known to be dependent on ammonium for their growth.

The total area of the human small intestine is 300-600 m2, stomach 1-2 m2, and large intestine 10-20 m2 and population of the enterocytes dominate over other cells in the gut. Enterocytes are the only cell in the gut where absorption of elementary nutrients such as amino acids, peptides, fatty acids, glycerol, and mono and disaccharides occur. In stomach and colon only water can be absorbed via the epithelial cells. The small bowel epithelium can also produce secretes for intestinal digest. Epithelial cell physiological life span is 72 h. Lectins, e. g. PHA can accelerate enterocyte turnover by 300% shortening the life span of enterocytes to 24 h or less (Puzei et al., 1993, Eur. Clin. Nutr., 47 : 691-699).

The small intestine The luminal surface of the small intestine is composed of a single layer of columnar epithelium overlaying mucosal projection, i. e. villi, which serve to enhance the surface area of the gastrointestinal tract.

The intestinal epithelium has two conflicting functions : it must serve as a protective barrier against luminal bacteria and toxins yet also absorb solutes necessary to maintain the well being of the host (Jankowski et al., (1994) Gut, 1 : 1-4). The conflict is notable at the intercellular space, which acts as a sieve that theoretically allows passage of selected solutes and water (Pappenheimer, J. R., (1990).

Gastrointestinal Liver Physiology 22 : 290-299 ; Soergel, K. H., (1993) Gastroenterology 105 : 1247-1250.) but prevents passage of bacterial toxins (Madara, J. L., (1990) American Journal of Pathology 137, 6, 1273-1281.), which, in turn, fluctuates in size depending on the degree to witch the attached cytoskeleton contracts (Madara et al., (1987) American Journal of Physiology - Cell Physiology 253 : 854-861). Intestinal injury, such as that caused by intestinal ischemia or inflammatory cell infiltration, results in enlargement of the intercellular space, initially caused by loosening of tight junction (Madara, J. L, (1989). l of Clin. Invest.

83 : 1089-1094) and subsequently caused by disruption of epithelial continuity (Madara, J. L., (1990) Pathobiology of the Intestinal Epithelial Barrier, American J. ofPathol. 137, 6, 1273-1281).

Alpha-ketoglutaric acid Glutamin and its derivatives e. g., alpha-ketoglutaric acid (AKG) are molecules which have a central role in the systemic and gut metabolism via Krebs cycle. However, the mechanisms are still not fully understood (Pierzynowski, S. G., and Sjödin, A. (1998) J. Anim. a. Feed Sci. 7 : 79-91 ; and Pierzynowski S. G., et al.

Eds : KBK Knutsen and J-E Lindberg., Uppsala 19-21 June, 2001).

AKG (2-oxo-pentanedioic acid, 2-oxoglutaric acid, alpha-oxoglutaric acid, alpha-oxopentanedioic acid, 2-Ketoglutaric acid, 2-oxo-1, 5-pentanedioic acid, 2- oxopentanedioic acid, 2-oxo-glutaric acid) can theoretically be a product of glutamin, glutamate, glutamic acid degradation in body metabolism. It may also serve as a precursor not only for glutamin and arginin, but also for some other amino acids, and are thus regarded as a protein catabolic protector. Olin at al., 1992 showed that when AKG was added to fish feed, urea emission was reduced.

Similarly, in humans were AKG is added to TPN solutions mixed with others amino acids, good protection of nitrogen loss after surgery is observed (Pierzynowski, S. G., and Sjödin, A. (1998) J. Anim. a F eed Sci. 7 : 79-91). In the case with the humans, the AKG is probably integrated with muscle protein degradation to serve requirements of intestinal tract during so-called postoperative stress, e. g. catabolism, starvation, etc.

The requirements of glutamin family related metabolites for gut function was proven recently by Reeds et al., (1996, am. J. of Physiol. -Endocrinology and Metablism 270 : 413-418) who reported almost 100 % glutamate/glutamine utilisation in the first pas in infant pigs small intestine.

AKG can be an important energy donor via few transformation pathways e. g. via ornithine and putrescine to GABA or succinate. Theoretically, AKG can also work as an ammonium ion scavenger possibly via transformation to glutamate/glutamine.

It is recognised-but never published-that enterocytes are dependent of their growth from ammonium.

It is thus highly desirable in the light of the aforementioned problems to develop means and methods for treating and improving mammals, such husbandry animals and humans, ability to adapt to e. g. weaning and thus further improve the growth and health of the mammal, which can also avoid the problems associated with the prior art means and methods. Also, there is a need for increasing the well being in renal patients. In this respect, the present invention addresses those needs and interests.

SUMMARY OF THE INVENTION In view of the foregoing disadvantages known in the art concerning dialysis relating to renal failure or early weaning in a vertebrate, such as a mammal, the present invention provides a new composition, use and methods for obtaining a mature gastrointestinal tract (GIT), quicker protein synthesis via enterocytes in the GIT and less nitrogen evacuation via the kidney in the form of urea in a vertebrate, such as a mammal.

An object of the present invention is to provide a composition comprising phytohaemagglutinin (PHA), and glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof.

Such a composition is a composition wherein the glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof are selected from the group consisting of glutamin, glutamate, alpha-ketoglutaric acid (AKG), ornitine-AKG, arginine-AKG, glutamine-AKG, glutamate AKG, leucine-AKG and other salts of AKG with other amino acids and amino acids derivates ; mono-and di - metal salts of AKG such as CaAKG, NaAKG ; mono-and di-metal salts of glutamin or glutamate such as Ca-glutamine, Na-glutamate ; glutamin dipepetides and oligopeptides e. g., L-alanyl-L-glutamine, glycyl-L-glutamine and other peptides of glutamin with other amino acids ; glutamate dipeptides and oligopeptides e. g., glutamate-glutamine and other peptides of glutamate with other amino acids ; glutamate and glutamin polymers.

The present invention also provides a pharmaceutical preparation comprising the composition according to the invention, together with a pharmaceutically acceptable carrier and/or additives. Also, a coated preparation comprising the aforementioned composition is disclosed as well as a preparation wherein the composition is released by controlled release, such as a slow-release.

Still further, a food or feed supplement comprising the composition according to the invention, and/or additives. Such a food or feed supplement according to claim the invention, wherein the composition is a dietary supplement

and/or a component in fortin of solid food and/or beverage.

Furthermore, the invention provides a use of the composition according to the invention for the manufacture of a preparation for the prevention, alleviation or treatment of diarrhoea. The use may be wherein the diarrhoea is allergic in a premature or neonatal mammal.

Still furthermore the present invention relates to a use of the composition according to the invention, for the manufacture of a preparation for the treatment of immature gastrointestinal tract in a premature or neonatal mammal.

Even still further, the present invention relates to a method for obtaining a mature GIT in a premature or neonatal mammal, comprising administering to the mammal in the need thereof the composition according to the invention, or the pharmaceutical preparation according to the invention, or the food or feed supplement according to the invention.

Also, the present invention discloses a method for treatment, prevention or alleviation of renal failure or its early symptoms, the steps of administering to a mammal in the need thereof the composition according to the invention, or the pharmaceutical preparation according to the invention, or the food or feed supplement according to the invention.

SHORT DESCRIPTION OF DRAWINGS Fig. 1 shows photomicrograph of duodenum HCL treated (A) and AKG + PHA treated (B) rats. 1-thickness of tunica mucosa ; 2-crypts depth (objective x 2. 5), Fig. 2 shows immunohistochemical staining of cholecystokinin, CCK, positive cells in rats'duodenum Counterstaining with Mayor's hematoxylin. CCK positive cells are marked with arrows. A, B-HCL treated rat. C, D-AKG + PHA treated rat. A, C-objective x 20 ; B, D-objective x 100, Fig. 3 shows immunohistochemical staining of neuropeptide Y in rats' duodenum. Counterstaining with Mayor's hematoxylin. Neuropeptide Y contained neuronal cells marked by arrows. A, B-HCL treated rat. C, D-AKG + PHA treated rat. A, C-objective x 20 ; B, D-objective x 100, Fig. 4 shows urine nitrogen analysed day by day, from the control group treated with HCL. Rat no. 1 in A, no 2 in B, and no 3 in C, Fig. 5 shows urine nitrogen analysed day by day, from the group treated with PHA. Rat no. 9 in A, no 10 in B, and no 11 in C, Fig. 6 shows urine nitrogen analysed day by day, from the group treated with AKG. Rat no. 15 in A, no 16 in B, and no 17 in C, and Fig. 7. shows urine nitrogen analysed day by day, from the AKG + PHA treated group. Rat no. 21 in A, no 22 in B, and no 23 in C.

DETAILED DESCRIPTION Defìnitions The term"premature"is in humans intended to mean an infant that is born too early, i. e. one usually born after the twenty-seventh week and before full term, and arbitrarily defined as an infant weighing 1000-2499g at birth, having poor to good chance of survival, depending on the weight. In other mammals, it is intended to mean a progeny that is born too early, i. e. born before full term.

The term"neonatal"is intended to mean the first four weeks after birth in a human.

The term"mature gastrointestinal tract" (mature GIT) is herein intended to mean a GIT ready to digest, absorb and process all kind of non-milk or non-yolk dry food. Mature GIT is adaptive, which means that reversible changes can occur. A mature GIT is not permeable to macromolecules. Elementary dietary molecules, such as amino acids, monoglicerides, fatty acids, and calcium ions, iron ions, natrim ions, can easily penetrate the mature GIT mucosa or be absorbed to eneterocytes.

The tenn"premature GIT"and the term"neonatal GIT"is herein intended to mean an immature GIT characterised in a predominat capacity of the GIT to digest milk, or yolk in birds. Such an immature GIT is characterised by low secretion of enzymes, long villies with high permeability to macromolecules, low bacteria load and mostly colonised with lactobacilli.

The term"level of digest"is herein intended to mean content of digest i. e. the amount of processed food in the GIT The term"level of digestion"is herein intended to mean a level of the enzymatic activity as well as other processes linked to digestion in the GIT lumen.

As used herein,"pharmaceutical composition"means therapeutically effective composition according to the invention.

A"therapeutically effective amount", or"effective amount", or "therapeutically effective", as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent ; i. e., a carrier, or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired

therapeutic effect in association with the required diluent ; i. e., carrier, or additive. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.

The term"derviate"or"derivative"is herein intended to mean a chemical substance derived from mother substance either directly or by modification or partial substitution.

The term"analogue"or analog"is herein intended to mean compounds that are structurally similar to another, but are not necessarily isomers. Analogs have similar function (s) but differ in structure or evolutionary origin.

As used herein,"treating", means treating for curing which may be a full curing or a partial curing of a condition or conditions.

The term"alleviate"is herein intended to mean not only a reduction of intensity of a condition or indication, but also postponing onset of a condition or indication.

The term"prevent"is herein intended to mean to ensure that something does not happen, e. g. that a condition or indication relating to an immature GIT does not happen. By preventing a certain condition or indication, the onset of such condition or indication is postponed.

A composition As revealed above, the present invention relates to means and methods for treating, alleviating or preventing any condition associated an immature GIT.

Accordingly, the present invention discloses a composition comprising phytohaemagglutinin (PHA), and glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof.

Glutamin derivate, metabolites, or analogues are e. g. alpha-ketoglutaric acid (AKG), and derivates, metabolites and analogues of AKG as described and exemplified in further detail below.

Specific embodiments may be wherein the glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof are selected from the group consisting of glutamin, glutamate, a-ketoglutaric acid (AKG), ornitine-AKG, arginine-AKG, glutamine-AKG, glutamate AKG, leucine-AKG and other salts of AKG with other amino acids and amino acids derivates ; mono-and di-metal salts of AKG such as CaAKG, NaAKG ; mono-and di-metal salts of glutamin or glutamate such as Ca-glutamine, Na-glutamate ; glutamin dipepetides and

oligopeptides e. g., L-alanyl-L-glutamine, glycyl-L-glutamine and other peptides of glutamin with other amino acids ; glutamate dipeptides and oligopeptides e. g., glutamate-glutamine and other peptides of glutamate with other amino acids ; glutamate and glutamin polymers in a composition according to the invention.

Even further, the composition may be a pharmaceutical preparation.

Still even further embodiments of the inventions are wherein the composition is a dietary supplement and/or a component in form of solid food, powder, flakes or granule format or in a beverage.

The composition needs to be administered in certain amounts to a mammal in the need thereof. Such amounts may dependent on the bodyweight of the mammal.

Accordingly, the dosage of the composition is related to the amount administered.

As for the administration of a composition to an mammal, such as a human being, all ages of mammals are contemplated, as is further exemplified in the experimental part below such as Example 3 which is performed in older rats.

The composition further comprises PHA, a lectin of the legume lectin group.

PHA is unique in its specificity to complex oligosaccharide chains. The choice of lectin is important for the invention to work in a desired manner. Other lectins than PHA with other specificities, such as chitin-binding lectins, e. g., lectins derived from Solanum tuberosum with a specificity to N-acetylglucoseamine, will, thus, not work in a similar manner as PHA.

For PHA, the amount may bel pg-500 mg/kg bodyweight as a daily dose.

Specific embodiments uses PHA in an amount of 10-150 mg/kg bodyweight, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mg/kg bodyweight, as a daily dose. This high dosage may be used for obtaining a mature GIT or an accelerated enterocyte turnover in a short time treatment, such as in piglets before weaning.

Further embodiments uses PHA in an amount of 50-500 pg/kg bodyweight, such as 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or 500 Fg/kg bodyweight, as a daily dose. Such a lower dosage is more used in a long term, e. g. several years, treatment, e. g. as for prevention, alleviation or treatment of renal failure.

Similarly, the composition according to the invention contains glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof in an effective amount for a mammal in the need thereof. Such amounts are accordingly dependent on the body weight of the mammal. Different embodiments of the invention therefore includes a composition wherein glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof, is in amount of is in an amount of 0. 01-0. 5 g/kg bodyweight, such as 0. 01, 0. 05, 0. 1, 0. 15, 0. 2, 0. 25, 0. 3, 0. 35, 0. 4, 0. 45, or 0. 5 g/kg bodyweight, per daily dose.

Still further, the amount of glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof may be in an amount of 0. 01-0. 2 g/kg bodyweight per daily dose, such as 0. 01, 0. 02, 0. 03, 0. 04, 0. 05, 0. 06, 0. 07, 0. 08, 0. 09, 0. 1, 0. 11, 0. 12, 0. 13, 0. 14, 0. 15, 0. 16, 0. 17, 0. 18, 0. 19, or 0. 2 g/kg bodyweight per daily dose.

Even further embodiments uses glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof in an amount of 0. 01-0. 1 g/kg bodyweight per daily dose, such as 0. 01, 0. 02, 0. 03, 0. 04, 0. 05, 0. 06, 0. 07, 0. 08, 0. 09, or 0. 1 g/kg bodyweight per daily dose.

Additives to the composition The composition according to the invention may, of course in different embodiments contain relevant additives, such as electrolytes, fatty acids and amino acids. Other relevant additives are excipients, which are acceptable and compatible with the active ingredients, e. g. PHA and glutamin, glutamin derivates or metabolites, glutamin analogues or mixtures thereof. Suitable excipients are, for example, water, saline, dextrose, sucrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH, buffering agents, which may enhance the effectiveness of the active ingredient.

In even further embodiments, the composition may include other relevant additives, such as filings and various buffers, (e. g., Tris-HCI., acetate, phosphate) ; to set a fix pH and ionic strength, and/or additives such as albumin or gelatine to prevent absorption to surfaces, detergents (e. g., Tween 20, Tween80, Pluronic F68, bile acid salts), solubilizing agents (e. g., glycerol, polyethyleneglycerol), anti- oxidants (e. g., ascorbic acid, sodium metabisulfite), preservatives (e. g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e. g., lactose, mannitol, sucrise).

Even further embodiments include covalent attachment of polymers such as polyethylene glycol to the composition, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts.

Medical use of the composition The composition may, according to the invention, be a composition for medical use. As such, the medical use includes medical use for animals, preferably mammals, i. e. veterinary use. Also, the medical use is a use in a human being in the

need thereof.

A pharmaceutical preparation The invention also discloses a pharmaceutical preparation comprising the composition according to the invention, together with a pharmaceutically acceptable carrier and/or additives.

The pharmaceutical preparation according to the invention, may be together with a pharmaceutically acceptable carrier and/or additives, such as diluents, pre- servatives, solubilizers, emulsifiers, adjuvants and/or carriers useful in the pharma- ceutical preparation disclosed in the present invention. Such pharmaceutically acceptable carrier and/or additives are known to the skilled man in the art.

Further, as used herein"pharmaceutically acceptable carriers"are well known to those skilled in the art and may include, but are not limited to, 0. 01-0. 05M phosphate buffer or 0. 8% saline. Further, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, cheating agents, inert gases and the like, as well as, but not limited to, other additives mentioned in the paragraph "Additives to th e composition"above. The amount and dosages in a pharmaceutical compositions is mentioned in the paragraph "the composition" above.

A food or feed supplement The invention also discloses a food or feed supplement comprising the composition or the pharmaceutical composition according to the invention, and/or additives and supplements described above.

Specific embodiments of the invention are wherein the composition is a dietary supplement and/or a component in form of solid food and/or beverage.

Administration of the food or feed supplement may be performed in different ways depending what species of animal, such as a mammal, e. g. a pig, rat or a human being, to treat, on the condition of the vertebrate in the need thereof, and the specific indication to treat, prevent or alleviate, e. g. kidney failure, early weaning symptoms or diarrhoea.

In one embodiment, food or feed supplement is a dietary supplement and/or a component in the form of solid food and/or beverage. Further embodiments may be in suspensions or solutions, such as a beverage further described below. Also, the

formats may be in capsules or tablets, such as chewable or soluble, e. g. effervescent tablets, as well as a powder, e. g. water soluble powder, flakes, granules or other dry formats known to the skilled man in the art, such as pellets, e. g. as micropellets, and grains.

The administration may be as a rectal or oral food or feed supplement.

The food and feed supplement may also be emulsified. The active therapeutic ingredient may then be mixed with excipients, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH, buffering agents, which may enhance the effectiveness of the active ingredient.

Different formats of the food or feed supplement may be supplied, such as solid food, liquids or lyophilized or otherwise dried formulations. It may include diluents of various buffers (e. g., Tris-HCI., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatine to prevent absorption to surfaces, detergents (e. g., Tween 2Q, Tween80, Pluronic F68, bile acid salts), solubilizing agents (e. g., glycerol, polyethyleneglycerol), anti-oxidants (e. g., ascorbic acid, sodium metabisulfite), preservatives (e. g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e. g., lactose, mannitol).

Still further embodiments include covalent attachment of polymers such as polyethylene glycol to the composition, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts.

A beverage In one embodiment, the food or feed supplement is administered in the form of a beverage, or a dry composition thereof, in any of the methods according to the invention.

The beverage comprises an effective amount of the composition or the pharmaceutical composition described above, together with a nutritionally acceptable water-soluble carrier, such as electrolytes, vitamins, carbohydrates, fat and proteins. All of these components are supplied in a dried form if the beverage is provided in a dry form. A beverage provided ready for consumption further comprises water. The final beverage solution may also have a controlled tonicity and acidity, e. g. as a buffered solution according to the general suggestions in the paragraph above.

The pH is may be in the range of about 2-5, and in particularly about 2-4, to prevent bacterial and fungal growth. A sterilised beverage may also be used, with a pH of about 6-8.

The beverage may be supplied alone or in combination with one or more therapeutically effective compositions, such as other formats of the food or feed supplement described above.

A controlled release preparation In one embodiment, the composition according to the invention is administered in the form of a preparation enabling a controlled release, such as a slow-release, of the composition to achieve a desired release. One desired release according to the invention may be e. g. in the small intestine instead of the stomach.

In one embodiment, the controlled release, such as a slow-release, prepara- tion is in the format of a tablet, granules and matrix granules in solid food or a beverage, powder, such as a water soluble powder, flake format, grains or a capsule.

Such a preparation may be a coated preparation, where the coating may enable the slow release of the composition.

The administration of a controlled release preparation may be according to any of the methods discloses in the invention.

Use of the composition The use of the composition according to the invention may be for the manufacture of a preparation for the prevention, alleviation or treatment of diarrhoea. Such a diarrhoea may be an allergic diarrhoea, but may also be a diarrhoea due to general low functioning of the GIT, e. g. as a low liquid absorption of the intestine.

The allergic diarrhoea may in specific embodiments be diarrhoea in a premature or neonatal mammal. Such a premature or neonatal mammal may in different embodiments be a human being. Other embodiments are wherein the premature or neonatal mammal may be a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm animals, a dog, a cat and other pets.

The described use may, according to the invention, also be a use wherein the preparation is a pharmaceutical preparation. Other embodiments are wherein the preparation is a dietary food or feed supplement and/or a component in form of solid food and/or beverage, as disclosed in the invention.

Treatment of immature gastrointestinal tract The invention also discloses the use of a composition according to the

invention, for the manufacture of a preparation for the treatment of in a premature or neonatal mammal.

Specific embodiments are wherein the premature or neonatal mammal is a human being. Other embodiments are wherein the premature or neonatal mammal is a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm animals, a dog, a cat and other pets.

The described use may, according to the invention, also be a use wherein the preparation is a pharmaceutical preparation. Other embodiments are wherein the preparation is a dietary food or feed supplement and/or a component in form of solid food and/or beverage, as disclosed in the invention.

As for a pig, or a piglet, before weaning, the treatment may be one, two, three, four, five or even ten time treatment for 5-7 days before weaning.

As for a human, with an immature GIT, the dose regime may be decided individually, but the dose regime may be similar, but not limited to, the dosages as for a pig.

Treatment of an infection The invention also discloses a use of the composition for the manufacture of a preparation for the treatment, prevention or alleviation of an infection in a premature or neonatal mammal. Such a use, may allow for less use of widely use antibiotics in the treatment of infections in premature or neonatal mammals, such as neonatal pigs or premature children, which is highly beneficial.

The infection may be of bacterial origin, but may also be of viral origin or a combination thereof.

The described use may, according to the invention, also be a use wherein the preparation is a pharmaceutical preparation. Other embodiments are wherein the preparation is a dietary supplement and/or a component in form of solid food and/or beverage.

Treatment, prevention or alleviation of renal failure The invention also discloses a use of the composition according to the invention, for manufacture of a preparation for the treatment, prevention or alleviation of renal failure. To alleviate the renal failure will postpone the onset of an active treatment. One example is where the composition according to the invention administered to an uremic patient, whereby the predialysis phase may be prolonged, i. e. an active treatment using dialysis is postponed or prevented.

There are two forms of renal failure, acute and chronic renal failure (ACF and CRF). Acute renal failure can normally be reversed, while chronic normally progresses. In different embodiments of the invention, all of those mentioned stages

of renal failures may be treated, prevented or alleviated by the composition according to the invention. The age of the mammal treated, such as a human being, is not limiting the invention. As such, all ages are contemplated.

In one embodiment, the renal failure is an increased level of blood nitrogen in the form of urea. Renal failure may be treated by dialysis of the patient's blood, a procedure which is both time consuming and costly, or transplantation. By using the composition according to the invention such procedure may be unnecessary or may not have to be performed as frequent as normally. Also, it may shorten the time spent in dialysis for the patient in the need thereof. As such, the life quality and the well being of the patient is increased. Also, the cost of treating dialysis patients is reduced.

In specific embodiments, the species is a mammal. Even further, the mammal may be a human being. Other embodiments arc wherein the premature or neonatal mammal is a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm animals, a dog, a cat and other pets.

As for renal failure,. the treatment may be life-long treatment, as a supple- ment, or substituting completely or partially the regular dialysis procedure or trans- plantation when having a renal failure.

In specific embodiments, uremic patients are treated with the composition or the pharmceutical composition according to the invention. Treating such patients with the composition or the pharmaceutical composition according to the invention, may delay or prevent the onset of an active treatment, e. g. the dialysis or trans- plantation. It may also increase the life quality and well being of the patient, due to less medical care. Also the cost when treating such a patient with the composition according to the invention may be decreased since administering of the composition may be performed by the patient itself if a human being, by a caretaker if a young patient, or an animal caretaker if a mammal such as a horse, rat or pig.

Treatment of predialysis patients may postpone the active treatment, i. e. dialysis or transplantation, for several years.

A method for obtaining a mature GIT Also disclosed in the invention is a method for obtaining a mature GIT in a premature or neonatal mammal, comprising administering to the mammal in the need thereof the composition or the pharmaceutical preparation according to the invention, or the food or feed supplement according to the invention. Such a method may in different embodiments be a method, wherein the premature or neonatal mammal is a human being. Further embodiments comprise the method according to the invention wherein the premature or neonatal mammal is a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm

animals, a dog, a cat and other pets.

Still even further embodiments of the method are wherein the administering to the mammal in the need thereof is nasal, such as via an inhalator, oral or rectal as disclosed in the invention.

A method for modulating GIT The invention also discloses a method for modulating GIT in a premature or neonatal mammal, comprising administering to the mammal in the need thereof the composition according to the invention, or the pharmaceutical preparation according to the invention, or the food or feed supplement according to the invention.

Such a method may in different embodiments be a method, wherein the premature or neonatal mammal is a human being. Further embodiments comprises the method according to the invention wherein the premature or neonatal mammal is a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm animals, a dog, a cat and other pets.

Still even further embodiments of the method are wherein the administering to the mammal in the need thereof is nasal, such as via an inhalator, oral or rectal as disclosed in the invention.

A method for prevention, alleviation or treatment of diarrhoea The invention also includes a method for prevention, alleviation or treatment of diarrhoea comprising the steps of administering to the mammal in the need thereof the composition according to the invention, or the pharmaceutical preparation according to the invention, or the food or feed supplement according to the invention.

Such a method may in different embodiments be a method, wherein the premature or neonatal mammal is a human being. Further embodiments comprise the method according to the invention wherein the premature or neonatal mammal is a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm animals, a dog, a cat and other pets.

Still even further embodiments of the method are wherein the administering to the mammal in the need thereof is nasal, such as via an inhalator, oral or rectal as disclosed in the invention.

Such a diarrhoea may be an allergic diarrhoea, but may also be a diarrhoea due to general low functioning of the GIT, e. g. as a low liquid absorption of the intestine.

A method for the treatment, alleviation or prevention of an infection The invention also discloses a method for the treatment, alleviation or

prevention of an infection in a premature or neonatal mammal comprising the steps of administering to the mammal in the need thereof the composition according to the invention, or the pharmaceutical preparation according to the invention, or the food or feed supplement according to the invention.

Such a method may in different embodiments be a method, wherein the premature or neonatal mammal is a human being. Further embodiments comprise the method according to the invention wherein the premature or neonatal mammal is a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm animals, a dog, a cat and other pets.

Still even further embodiments of the method are wherein the administering to the mammal in the need thereof is nasal, such as via an inhalator, oral or rectal as disclosed in the invention.

A method for gaining body weigh in a premature or neonatal mammal The invention also discloses a method for gaining body weigh in a premature or neonatal mammal comprising the steps of administering to the mammal in the need thereof the composition according to the invention, or the pharmaceutical preparation according to the invention, or the food or feed supplement according to the invention.

Such a method may in different embodiments be a method, wherein the premature or neonatal mammal is a human being. Further embodiments comprises the method according to the invention wherein the premature or neonatal mammal is a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm animals, a dog, a cat and other pets.

Still even further embodiments of the method are wherein the administering to the mammal in the need thereof is nasal, such as via an inhalator, oral or rectal as disclosed in the invention.

A method for treatment, prevention or alleviation of renal failure The invention also discloses a method for treatment, prevention or alleviation of renal failure, the steps of administering to the mammal in the need thereof the composition according the invention, or the pharmaceutical preparation according to the invention, or the food or feed supplement according to the invention.

The method may be a method wherein the patient is a human being. Other embodiments include a method wherein the mammal is a rodent, such as a mouse, rat, guinea pig, or a rabbit ; a cow, a horse, a pig or piglet and other farm animals, a dog, a cat and other pets.

The mammal, such as a human being, may be an elderly human being often in the need of dialysis due to renal failure due to ageing. Still, younger mammals,

such as a human being, may also be in the need for dialysis due to an early renal failure. This may be caused by an injury of the mammal, such as a human being, or a defect that the individual is born with. In all mentioned cases, the method according to the invention may be used as a complement or instead of regular dialysis performance.

According to the method for treatment, prevention or alleviation of renal failure, the composition according o the invention is administered. The administra- tion may be nasal, such as via an inhalator, oral or rectal as disclosed in the inven- tion.

EXPERIMENTAL PART The influence of PHA and AKG on enetrocyte turnover, intestine maturation and urea synthesis in the ornithine cycle.

Objective Two experiments, experiment 1 and 2, were conducted to evaluate the effect of PHA and AKG on rat performance and nitrogen excretion pathways.

Animals used All rats came from the Sprague Dawley (E) outbreed Laboratory, M&B A/S, Denmark. Rats were caged individually in metabolic cages (60% humidity with 12 hours of light per 24 hours) and feed and water were provided ad libitum at Dept of Animal Physiology, Lund, Sweden.

Diet conditions Diet formulation is presented in table 1. Groups were equalised with regard to body weight. Feed, liquid, control as well as treatment drink consumption, and urine and faeces production was measured every day, while treatments with PHA, control or water were conducted every second day. Treatments and experimental conditions were conducted according to the recommendations of the Federation of the European Laboratory Animal Science Associations (FELASA) concerning the protection of experimental animals.

Table 1. Composition of diets.

Main Ingredients : Wheat, barley, Extracted Soya bean meal, Fat Blend, Electrolytes, Vitamin premixa, Amino Acids Calculated diet composition, % Dig. Crude Protein 17. 37 Dig. Crude Oil 3. 76 Dig. energy, MJ/kg 13. 75 Calcium 0. 65 Total Phosphorus 0. 51 Available Phosphorus 0. 24 Lysine 1. 26 Methionine 0. 30 provided the following per kilo of diet : Vitamin A, 14960 I. U ; Vitamin D3, 1574 I. U ; Vitamin E, 110. 4 mg ; Thiamin, 17. 6 mg ; Riboflavin, 13. 2 mg ; Pyrodoxine, 17. 9 mg ; Vitamin B12, 35 llg ; Vitamin K, 19. 9 mg ; Folic Acid, 3. 1 mg ; Nicotinic Acid ; 34. 1 mg ; Panthotenic Acid, 34. 1 mg ; Choline, 1557. 0 mg ; Ionsitol, 1811.4 mg ; Biotin, 369. 2 µg.

Experiment 1 Animals used Twentyone males and two females, seven weeks old, were randomly divided into 4 experimental groups : 1) Control 2) HCL + PHA 3) AKG 4) AKG + PHA 5 animals were in the control group and 6 in each of the treatments.

Treatment The treatment with HCL was provided through water application. Also, the control group received HCL.

AKG was applied through water, while PHA was given by stomach tube.

The stock solution of PHA Crude (20%) was in 0. 9% NaCl in water : 50mg PHA/mL, 20 mL/kg bodyweight.

Rats receiving water by stomach tube were also dosed 20 mL/kg bodyweight.

Rats were in trial for 9 days. Initially body weight of the rats at trial start was 252, 6g <BR> <BR> <BR> zL lg.<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>Experiment 2 Animals used Twenty three, seven weeks old, male rats, were randomly divided into 4 experimental groups 1) Control 2) HCL pH 4. 6 3) PHA 4) Electrolytes, pH 7 5 animals were used in the control group and 6 in each treatment.

Treatment The water, HCL and electrolyte treatment was provided through water application.

PHA was given by stomach tube. The stock solution of PHA Crude (20%) was in 0. 9% NaCI in water : 50mg PHA/mL, 20 mL/kg bodyweight.

Rats receiving water by stomach tube, for identical treatment, were also dosed 20 mL/kg bodyweight. Rats were in trial for 11 days.

Initially body weight of the rats at trial start was 236, 0 g 1, 4 g. <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>Experiment 3<BR> <BR> <BR> <BR> <BR> Objective In this particular experiment, older rats were used. A rat age of 50-60 weeks corresponds approximately with a human age of 70-75 years. The objective is to show that even in an older animal the accelerated enterocyte turnover will, as well as in a young animal, give an increased protein turnover in the intestine.

Animals used 23 rats, 12 males and 12 females all at the age of 50-60 weeks were used. The weight was about 300-smog per rat dependent on sex. The rats were randomly divided into 4 experimental groups 1) Control 2) AKG 3) AKG + PHA 100% 4) AKG + PHA 1%

Treatment During treatment, one group of 5 animals and 3 x 6 animals are kept for 3 weeks in normal single animal cages and then put in metabolic cages for 6-7 days.

Treatment is taken place during the entire period of 4 weeks.

Group 1 : Water is enriched with electrolytes and 0. 1M HCL, pH 4. 6. Ha0 is given each second or third day by stomach tube (20 ml/kg bwt.).

Group 2 : Water is enriched with electrolytes and 0. 1 M AKG. H2O is given each second or third day by stomach tube (20 ml/kg bwt.).

Group 3 : Water is enriched with electrolytes and 0. 1M AKG + PHA (Crude) is given each second or third day by stomach tube (PHA-1000 mg/kg bwt., in 0. 9% NaCl - 20 ml/kg bwt.) Group 4 : Water is enriched with electrolytes and 0. 1 M AKG + PHA (Crude) is given each second or third day by stomach tube (PHA-10 mg/kg bwt., in 0. 9% NaCl - 20 ml/kg bwt.).

Stock solution of crude (20%) PHA in water : 50mg/ml, 20 ml/kg bwt.

Urine and faeces sampling in Experiment 1-3 The collection of urine was performed every day in a cup with 10 mL of a 50% H2 SO 4 solution, to stop microbial activity.

Faeces were kept dry by collection into a cup.

The total level of urine was measured daily, and a pooled homogenised sample of 10 mL was collected for urea analysis. The weight of faeces was also measured on a daily bases, followed by collecting into 60 mL of 50% H2 SO 4 solution. At the end of the experiment the total collection of faeces was sent for nitrogen analysis.

Analysis of total nitrogen content, N, in Experiment 1-3 Nitrogen content was measured on a Leco Nitrogen and Protein Determinator Fop-428, Leco Corporation, St. Joseph, MI., U. S. A. The instrument was set up in low range mode. The urine samples, 100-200µl, were analysed in tin capsules 15x 6mm.

The faeces samples, 150-200mg, were analysed in tin foil cups. A glycine standard solution containing 1% nitrogen was used as reference. For all nitrogen determinations, analysis was performed on duplicate samples.

Prior to analysis, the faeces samples were homogenised in a Sorvall Omni- Mixer homogeniser. To obtain pooled urine samples for nitrogen determination aliquots of 1% of the daily urine volume were pooled.

Sampling of GI-tract and digest for histopathological analysis in Experiment 1-3 Digest contents of stomach, proximal, distal small intestine, cecum and colon was weighed and measured for AKG concentration.

The duodenum of the animal was fixed in Bouin's solution, routinely dehydrated and embedded in paraffin. Paraffin sections (5 pLm) were cut and mounted on Super Frost/Plus slides (Histolab, Sweden). For the histological study under a light microscope a staining with hematoxylin and eosin was used. In the duodenum preparations the crypt depth, tunica mucosa thickness and the epithelial cells height and basal part width were measured.

From each animal 40 well oriented villi and crypt lying outside the area with Peyer's patches were measured at low magnification (objective x 2. 5). Tunica mucosa thickness was measured as a distance from the muscularis mucosa to the tip of villus, whereas crypt depth was measured from the muscularis mucosa to the level of the crypt opening as seen in figure 1). The enterocytes height of 10 well- oriented villi were measured at high magnification (objective x 100). One thousand cells were examined for each animal.

The immunohistochemical reaction was performed with monoclonal antiserum versus neuropeptide Y (Sigma, Sweden) or cholecystokinin (CCK) (Sigma, Sweden) in a dilution of 1/8000. An indirect avidin-biotin-peroxidase method was applied with using Histomouse SP Bulk kit Broad Spectrum (Zymed, USA).

Peroxidase was visualised using 3-amino-9-ethylcarbazole as a chromogen and the sections were lightly counterstained with hematoxylin according to the manufacturer's instructions. All sections were stained at the same time. The specificity of staining was controlled by substituting the primary antibodies with a buffer. Immunoreactivity was eliminated by such procedure.

Calculations and statistics in Experiment 1-3 Total feed intake was calculated from the level of used feed per day, but an approximation was made to compensate for the feed which went into the cup collecting faeces, by actually measuring the feed content before removing the faeces. This correction factor was found to be constant for all treatments and amounted to 10. 77% of the total feed used. The final feed intake has therefore been adjusted by this factor. The nitrogen level in feed is calculated from the level of protein in feed, which is 19. 76%.

Data have been analysed by students't-test. The comparisons of each variable in the histopathological analysis were done by performing a one-way ANOVA. When the overall ANOVA was significant, we then performed pair wise

comparisons with t-test. In statistical analyses P<0. 05 was taken as the level of significance.

Results of Experiment 1-3 Experiment 1 Weight In experiment 1 the start weight in the control group was on average 252. 6 g/rat, and the weight in the treated groups were 250. 38-250. 76, without significant difference. The results on the final weight show, that the PHA + HCL treatment gave a reduced bodyweight, P< 0. 07, and the AKG + PHA treatment gave a reduced bodyweight, P< 0. 06 compared to the control group. The final growth was found to be significantly reduced, P< 0. 03 and P< 0. 05 for the same two treatments, compared to the control group.

Feed and water intake Feed intake was significantly reduced P<. 05 in the PHA + HCL treatment.

The AKG treatment was the only treatment, which actually increased its final weight, growth and feed intake compared to the control group.

The total water intake was reduced in all treatments compared to the control group, but only in the AKG + PHA treatment, it was reduced significantly, P<. 01.

The AKG treatment only showed a tendency of significance P<. 06 (Table 2).

Table 2. Performance and urine and faeces results per day per animal.

Trial Groups Daily Feed intake Water Urine, Faeces, g gain, g (Corr.), g Intake, mL mL HCL 5. 4 22. 4 48. 0 31. 9 11. 70 PHA + 3. 6*** 19. 0* 36. 4 18. 5 11. 68 <BR> <BR> <BR> HCL<BR> <BR> <BR> Experiment 1<BR> AKG 6.0 23.6 33.4 16.1** 11.58 AKG+ 3.2* 19.8 25.4***** 10.7*** 11.39 PHA * Water 5. 5 25. 0 23. 6 10. 1 12. 90 HCL 4. 8* 22. 9 52. 3* 33. 1** 11. 20 Experiment 2 PHA 2. 9** 20. 5** 21. 2 8. 9 11. 90 electrolytee 5. 1 21. 9 48. 8** 30. 6** 11. 40 5 *P<.05 ** P<. 04 ***P<.03

****P<.02 *****P<.01 Urine and faeces excretion A significant reduction in the level of urine excretion from both AKG treatments on P<. 04 and P<. 02 from the AKG and AKG + PHA, respectively. There was no difference between treatments with respect to the final level of faeces excreted after 9 days of trial. <BR> <BR> <BR> <BR> <BR> <BR> <P>Experiment 2<BR> <BR> <BR> <BR> Weight In experiment 2 the start weight in the control group was on average 237. 6 g/rat, while the weights in the treated groups were 234. 5-237. 1 g, without significant difference. The results on the final weight show, that the PHA treated rats was reduced P< 0. 06, compared to the control group. The total growth was found to be significantly reduced, P< 0. 05, and P< 0. 04 in the HCL and PHA treatments, compared to the control group.

Feed and weater intake Feed intake was significantly reduced P< 0. 04 in the PHA treatment, and P< 0. 06 in the electrolyte treated group. Total water intake was increased P< 0. 05 and P< 0. 04 in treatments HCL and Electrolytes, respectively (Table 2).

Urine and faeces excretion The level of urine was increased P< 0. 04 in both the HCL and in the electrolyte treated rats, while there were no difference in the total level of excreted faeces between the groups (Table 2).

Nitrogen excretion (urine and faeces) in experiment 1 and 2 The level of N excretion from urine is reduced. Both PHA treatments gave reduced levels, P< 0. 002 and P< 0. 001, in the PHA + HCL and AKG + PHA group, respectively. In experiment 2 the PHA treatment was also significantly reduced, P< 0. 04, compared to the control group of water treatment.

Non of the results on faeces in both experiments proved any difference as compared to the control groups, on the level of N in faeces (Table 3).

Conclusion in nitrogen measurements in Experiment 1-2 The relative level of nitrogen from urine and faeces, compared to HCL as control, group gives a clear indication of PHA as a factor able to divert the existing

relation between the nitrogen level in urine and faeces. In experiment 1, the level of nitrogen in urine and faeces is between 0. 68-0. 98 in the PHA + HCL treatment, while the AKG + PHA treatment gives a relation of 0. 67-1. 07. The same result is found in experiment 2, where the PHA treatment, have a relation of 0. 82-1. 12 (Table 3).

Table 3. Nitrogen excretion from urine and faeces per day, total N per day, and relative values of N from urine and faeces (g).

Trial Groups N in urine, N in faeces, Total Relative N- g/d g/d N/g U : F HCL 0. 38 0. 14 0. 52 1. 00 : 1. 00 PHA+ 0. 26** 0. 14 0. 40 0. 68 : 0. 98 <BR> <BR> <BR> HCL<BR> <BR> <BR> ERxperiment 1<BR> <BR> AKG 0.33 0.15 0.48 0.88:1.01 AKG+ 0.25*** 0.15 0.41 0.67:1.07 PHA Water 0. 37 0. 13 0. 50 1. 02 : 1. 03 HCL 0. 36 0. 16 0. 49 1. 00 : 1. 00 Experiment 2<BR> PHA 0.30* 0.14 0.44 0.82:1.12 Eledtrolytes 0.37 0.13 0.50 1.04:1.00 * P<. 04 ** p<. 002 ***P<. 001 Weight and digest from GI-tract in experiment 1-2 The weight of the GI-tract showed significant results in the small intestine and large intestine in experiment 1. Both PHA treated groups, PHA + HCL and AKG + PHA increased the weight, P< 0. 003 and P< 0. 006, respectively, in the small intestine, while only the AKG + PHA treatment increased, P< 0. 006, in the large intestine.

In experiment 2, the PHA treatment is reduced the weight, P< 0. 04, while the HCL treatment show a tendency of weight reduction, P< 0. 09, both compared to water in the control group. In cecum the PHA and the electrolyte treatments give weight reduction to a significant value, P< 0. 06, and the total GI-tract weight is reduced, P< 0. 04, in case of the HCL treatment (Table 4).

Table 4. Gastro Intestinal tract (GIT) weight, g.

Trial Groups Stomach, Small Large Cecum, g Total GIT g Intestine, Intestine, weight, g g g HCL 1.5 6.9 1.2 1.3 10.8 PHA+ 1. 4 8. 7**** 1. 3 1. 3 12. 8 HCL Experiment 1 AKG 1.4 7.1 1.3 1.2 11.0 AKG+ 1.4 8.1*** 1.5* 1.4 12..3 PHA Water 1. 4 7. 1 1. 1 1. 5 11. 0 HCL 1. 2 6. 6 1. 1 1. 1 10. 0* Experiment 2<BR> <BR> PHA 1.2** 7.9 1.0 1.2* 11.2 Electrolytes 1.3 6.4 1.2 1.1* 10.1 * P<. 06 ** P<. 04 *** P<. 006 **** p<. 003 The level of digest from the stomach in the GI-tract in experiment 1 was significantly increased P<0. 001 in the PHA + HCL treatment.

The level of digest was also increased P<0. 03 after the AKG treatment in the large intestine. On a total level, the digest was increased P<0. 01 in the PHA + HCL treatment.

In experiment 2, the digest level from the stomach was reduced, P<0. 01, after electrolyte treatment, compared to the water control group. All digest levels were reduced significantly in the small intestine, P<0. 02, P<0. 002 and P<0. 001 after the HCL, PHA and electrolyte treatments, respectively. Only the electrolyte treatment reduced the total level of digest P<0. 04 in compared to the water control group (Table 5).

Table 5. Gastro Intestinal tract (GIT) weight of digest, g.

Trial Groups Stomach, g Small Large Total GIT Intestine, g Intestine, g weight, g HCL 2. 2 2. 4 6. 8 11.. 3 PHA+ 4.4****** 3.0 7. 9 15. 3*** <BR> <BR> <BR> <BR> HCL<BR> <BR> <BR> Experiment 1<BR> <BR> AKG 1.3* 2.7 8.38* 12.4 AKG+ 1.8 2.4 7.8 12.1 PHA Water 1. 4 2. 6 7. 9 11. 9 HCL 1. 0 1. 9** 6. 3 9. 2 Experiment 2 PHA 1. 3 1.5***** 7.8 10. 4 Electrolytee 0. 3**** 1. 1****** 6. 3 7. 7* s * P<0. 04 ** p<0. 03 ***P<0. 02 ****P<0.01 ***** p<0. 002 *****P<0.001 morphometric analysis The morphometric analysis of the duodenum of PHA treated animals from experiment 1 in PHA+HCL or PHA+AKG treatment, showed differences compared to the control group with regards to crypts depth (Table 6, Fig. 1). PHA in combination with HCL increased crypt depth more pronounced than with AKG.

AKG treated animals demonstrated tendency only to increase crypt depth (Table 6).

Increasing of the crypt depth of PHA treated animals could be due to increasing of mitotic activity of epithelial cells. No significant difference of the thickness of tunica mucosa was found in treated animals (Table 6).

Table 6. The crypts depth and thickness of tunica mucosa of the duodenum of rats, (means + SD,, um) Groups Crypts depth Thickness of tunica mucosa HCL 173. 9 ffi 18. 75 766, 5 ~ 59, 20 PHA+HCL 253. 4 i 19. 85** 732, 7 i 56, 73 AKG 197. 1 23. 54 732, 7 i 56, 73 AKG + PHA 241. 5 ~ 49.51* 711,0 ~ 35, 51

* p <. os **P<.001 Morphometric analysis of the height of enterocytes and basal length did not show difference between the control group and the AKG+PHA treated rats as shown in table 7.

Table 7. Influence of AKG + PHA treated rats on duodenum epithelial cells (villi), (means + SD, urn) Groups. Height of cells Basal length of 10 cells HCL 37. 4 i 7. 02 58. 7 i 11. 3 AKG + PHA 33.9 ~ 4.07 60. 0 ~ 4.08 Preliminary results of immunohistochemical tissue examination of the control group and the AKG+PHA treated animals demonstrated no difference in abundance of choleocystoknin (CCK) positive cells in tunica mucosa (Fig. 2). At the same time, the same rats demonstrated difference in contents of Neuropeptide Y (NPY) positive materials. In the control group, the animal expression of NPY presented in granules of neuronal cells of the submucosa parasympathetic ganglia and goblet cells of epithelia (Fig. 3). In contrast, the AKG+PHA treated animals, demonstrated expression of NPY in granules of neuronal cells of the submucosa parasympathetic ganglia and no expression in goblet cells (Fig. 3).

Results from experiment 3 Materials and methods The animals'stomach and proximal part of gastrointestinal tract were fixed in Bouin's solution, routinely dehydrated and embedded in paraffin.

Paraffin sections (5 urn) were cut and mounted on Super Frost/Plus slides (Histolab, Sweden). For the histological study under a light microscope staining with hematoxylin and eosin was used.

In the proximal preparations the crypt depth and tunica mucosa thickness were measured. From each animal 10 well-oriented villi and crypt lying outside the area with Peyer's patches were measured at low magnification (objective x10).

Tunica mucosa thickness was measured as a distance from the muscularis mucosae to the tip of villus, whereas crypt depth was measured from the muscularis mucosae to the level of the crypt opening. In the stomach preparations the tunica mucosa thickness was measured at low magnification (objective x10).

Statistical evaluation in Experiment 3 Comparisons of each variable were made by performing a one-way ANOVA.

When the overall ANOVA was significant, a pair-wise comparison with t-test was done. In statistical analyses P<0. 05 was used as the level of significance.

Results in Experiment 3 The common morphology of stomach under experimental conditions has changed.

Group 2 AKG Depth of stomach pits increased in comparison with the control animals.

Significant variability of morphology within animal's samples of this group was observed. Two animals from 6 treated animals demonstrated multiple dilation (enlargements) of stomach glands. Thickness of tunica mucosa did not changed in comparison with the control animals and the results are shown in table 8.

Group 3 AKG+PHA 100% This treatment leads to the oedema development of connective tissue of stomach tunica mucosa (4 animals from 6). Animal &num 13 did not demonstrate any difference in comparison with the control animals. Increasing of thickness of tunica mucosa on 10% was statistically significant in this group as shown in table 8.

Group 4 AKG+PHA 1% Dystrophy of stomach gland was observed in this treated group. Numerous picnotic nucleus and hyperchromic cells located in neck part of stomach glands.

Enterocytes of stomach pits were thinner that that in control. At the same time there are no morphological changes in stomach from animal &num 19 was observed. Increasing of thickness of tunica mucosa on 9% was statistically significant (Table 8).

Table 8 Thickness of tunica mucosa of the stomach of different treated rats, (means+SD, um) using F-test (ANNOVA) P<0. 001 Groups Thickness of tunica mucosa HCL 519. 6 i 65. 25 AKG 521. 0 ~ 45. 25 P=0. 390 PHA+AKG100% 574. 0 i 62. 33* P=0. 026 AKG+PHA 1% 570. 0 i 86. 09* P=0. 011 * Statistically significant difference according to the t-test in comparison with the HCL group.

Proximal part of the GIT in experiment 3 Histological examination of proximal part of gastrointestinal tract demonstrated that there is diffuse leukocyte infiltration of tunica mucosa in treated animals. Decreasing of villi length pronounced also in all experimental group. AKG treatment alone leaded to the desquamation of enterocytes. In same cases there were signs of epithelia replacing by connective tissue. Enterocytes located on villi top vacuolated in same animals from all treated group.

As in case with stomach examination, large individual difference between single animals was notes. In three animals from AKG treated group there were no morphological changes. In PHA+AKG 100% treated group two animals saved control morphology.

Morphometric analysis of the proximal part of gastrointestinal tract of various treated animals in any compositions showed differences in comparison with control animals with respect to crypts depth and tunica mucosa thickness as shown in table 9. However, PHA in combination with AKG 100% increased crypt depth more pronounced (45%) than in last group (AKG+PHA 1%) as shown in table 9.

A significant increase of the thickness of tunica mucosa was found in all treated animal groups as shown in table 9. The most pronounced change was found in the PHA+AKG 100% treated group as shown in table 9.

Table 9 The crypts depth and thickness of tunica mucosa of the proximal part of gastrointestinal tract of different treated rats, (means+SD, urn). For statistical analysis F-test (ANNOVA) was used for both parameters : P<0. 001 Groups Crypts depth Thickness of tunica mucosa HCL 170. 8 i 17. 65 709, 7 i 49. 15 AKG 194. 5 i 17. 66* 762. 0 i 56. 73* PHA+AKG100 247. 7 ~ 19.54** 848. 8 ~ 52,63** % PHA+AKG 1% 197. 7 47. 41**774. 1 ~ 33.41** *p<O. OS **P < 0.001 Conclusions in Experiment 1-3 The results in the above experimental trials show that it is possible to divert the excretion of nitrogen from the urine to the faeces, using PHA in combination with AKG. This will give an application for treatment of human renal patients with an increase in wellbeing by prolonging the predialysis period.

AKG+PHA treatment may also be used as a healing factor for damaged intestinal mucosa, post infection or antibiotic treatment. This is due to a positive effect of AKG and PHA on the GIT maturation and cell turnover. AKG+PHA may be also be used for post-medical treatment of destroyed GIT mucosa, due to a similar effect.

Also, in experiment 3 using older rats, the same effect was shown as in a young animal (experiment 1 and 2), i. e. an increase in the combined effect when using PHA and AKG. Even in older rats, an accelerated enterocyte turnover, giving an accelerated protein turnover, will give a further benefit to an old animal in the intestine.