NO871935L - PROCEDURE FOR TREATING CATABOLIC DYSFUNCTION. - Google Patents

Description

The present invention was supported by research grants from the National Institutes of Health, Trauma Center, Grant No. GM29327-05, and the United States Department of the Army, Contract No. DAMD-17-81-C-1201, which gives the United States Government certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This is a partial continuation of application no. 775,214, filed on 12 September 1985.

The present invention relates to a method for treating or preventing tissue destruction in an animal suffering from catabolic dysfunction.

Catabolic dysfunctions are the physiological conditions in which the breakdown of an anatomical structure occurs. Anatomical structures that are usually affected in this way are skeletal muscles and intestinal epithelium. Such catabolic activity often occurs after surgery, sepsis, burns, cancer chemotherapy, radiation therapy/injury, glucocorticoid therapy or, often, inadequate nutritional intake. Such catabolic dysfunctions are a major cause of death and disability and are characterized by abnormal glutamine metabolism.

Glutamine is a non-essential amino acid that can be synthesized by most tissues. Unlike most amino acids, glutamine has two amine groups, a cx-amino acid and an amide dd group. It is the presence of the amide group that enables glutamine to remove ammonia from the peripheral tissues of the body and transport nitrogen to the internal organs. In addition, it is common for tissues that remove glutamine from the circulation to use the carbon skeleton for energy.

Glutaminase and glutamine synthetase are the two most important enzymes involved in the regulation of glutamine metabolism. Glutaminase catalyzes the hydrolysis of glutamine to glutamate and ammonia, while glutamine synthetase catalyzes the synthesis of glutamine from glutamate and ammonia. While most tissues have both of these enzymes, usually one is more active than the other, depending on the particular tissue.

Glutamine synthesis and release occurs primarily in skeletal muscle and the brain. In turn, glutamine is consumed by such reproductive cells as fibroblasts, lymphocytes, tumor cells and intestinal epithelial cells. It is characteristic that these cells have high levels of glutaminase activity and low levels of intracellular glutamine. This fact may also be clinically significant for patients with large wounds, inflammation associated with infection or a gastrointestinal dysfunction that precludes normal enteral nutrition, since the desirable development of cells in these cell types and under these conditions may depend on the availability of sufficient amounts of glutamine.

In the stomach/intestinal tract, glutamine is used as respiratory fuel. The enteral administration of glutamine results in increased uptake of luminal glutamine by the intestinal mucosa followed by an immediate decrease in uptake of glutamine from the circulation. The gut's consumption of glutamine is balanced between these two sources of glutamine.

Most of the glutamine absorbed by the stomach/intestinal tract takes place via the epithelial cells that stimulate the villi of the small intestine. The glutamine metabolism that takes place in the small intestine constitutes the main energy source for the intestines and produces precursors for hepatic ureagenesis and gluconeogenesis by utilizing nitrogen and carbon from other tissues.

Evidence for glutamine's essential role in maintaining normal intestinal structure and function was provided by Baskerville et al., British Journal of Experimental Pathology, 61.: 132

(1980). These researchers lowered plasma glutamine concentrations to undetectable levels by infusing purified glutaminase into rhesus monkeys, marmosets, rabbits, and mice. As a result of this treatment, these animals exhibited vomiting, diarrhoea, villus atrophy, mucosal ulcers and intestinal necrosis.

Martin et al., (US patent 2,283,817) describes a preparation which contains glutamine and which is used as a detoxification agent instead of as a dietary supplement. In the patent, glutamine is synergistically combined with other amino acids to act directly on a toxin to prevent any harmful effect.

In Shive et al. (US patent 2,868,693) the patentees describe glutamine-containing preparations for the treatment of gastric ulcers.

Further evidence of the potential protective effect of glutamine blé"*shown by Okabe et al., Digestive Disease, 20: 66

(1975), who found that glutamine could protect against aspirin-induced gastric ulcers in humans.

This glutamine demand in the viscera may be even greater during critical illness, when it is known that glutamine metabolism in the small intestine is increased (Souba et al., Surgery, 94(2): 342

(1983)).

The nutritional needs of patients who cannot satisfactorily supplement themselves with nutrition are currently met by the administration of enteral or parenteral diets. Enteral diets are usually administered using small-diameter tubes placed through the nose into the gastric or duodenal areas, or by surgical implantation such as in gastrostomy or jujunostomy. The enteral mixtures currently available can be divided into four basic categories: elemental, polymeric, modular and modified amino acids.; These mixtures contain' glutamine. a normal individual and not to a patient suffering from a catabolic disease. •' " '-•'- '"■ ■'-" '- Elemental mixtures require minimal digestive effort and consist primarily of small peptides and/or amino acids, glucose- oligosaccharides and vegetable oil or medium chain triglycerides.

In polymeric mixtures, complex foodstuffs such as e.g. soy protein, lactalbumin or casein as a source of protein, maltodextrins or corn syrup solids as a carbohydrate source and vegetable oils or milk fat as a fat source.

Modular diets can be prepared by combining protein, carbohydrate or fat with a monomer or polymer mixture to satisfy particular nutritional requirements.

Mixtures that are composed of altered amino acid compounds are used primarily for patients with genetic defects in nitrogen metabolism or acquired disturbances in nitrogen accumulation, as the purpose here is to limit the patient's intake of certain amino acids that can be harmful.

Parenteral diets are usually administered intravenously. These intravenous fluids are sterile solutions consisting of simple chemicals such as sugars, amino acids and electrolytes, which can be easily assimilated.

The term "total parenteral nutrition" (TPN) is used to describe compositions for use in patients who have their entire dietary needs met intravenously. Formulations for total parenteral nutrition, unlike enteral formulas, do not normally contain glutamine. The absence of glutamine from parenteral formulations is caused in part by its instability at room temperature, and the resulting generation of ammonia and pyroglutamic acid. There has also been some concern regarding the generation of glutamic acid from glutamine due to the potential toxicity of glutamic acid as a neurotransmitter. However, these concerns do not appear to be justified. At a pH close to neutrality, glutamine breaks down very slowly (Souba, S.C.D. Thesis in Harvard Medical School Library, June 1984).

Total parenteral nutrition results in villus atrophy, a phenomenon that is generally reversible when oral nutrition is resumed. Since TPN mixtures lack glutamine, the body's need for this amino acid must be met by synthesis in body tissues.

In critically ill patients, net protein catabolism is associated with markedly decreased muscle glutamine stores (Askanazi et al., Annals of Surgery, 192: 78 (1980); Askanazi et al., Annals of Surgery, 191: 465 (1980)), decreased glutamine plasma levels (Askanazi et al., Annals of Surgery, 192: 78 (1980); Askanazi et al., Annals of Surgery, 191: 465 (1980)), and an expected increase in intestinal glutamine utilization (Souba et al. , Archives of Surgery, 120: 66

(1985); Souba et al., Surgery, 94(2): 342 (1983)). Glucocorticoids are known to increase glutamine consumption in the small intestine (Souba et al., Surgical Forum, 34: 74 (1983)).

None of the known investigations have shown that the breakdown of the skeletal muscle, the atrophy of the intestinal villi or other catabolic dysfunctions, which occur during total parenteral nutrition, can be prevented by the administration of high levels of glutamine.

In the invention, if an animal has, or is at risk of having, a catabolic dysfunction, a therapeutically effective amount of glutamine." This amount of glutamine is greater than that which normally occurs in the diet of healthy individuals. This increased amount of glutamine is necessary to compensate for the greater demand for glutamine that occurs during certain catabolic dysfunctions. In the absence of exogenous glutamine during these catabolic dysfunctions, glutamine would be formed by the breakdown: of muscle tissue. Decrease in plasma glutamine concentrations despite accelerated glutamine release from the muscle during catabolic dysfunctions, indicates systemic glutamine deficiency. Despite accelerated glutamine release from the muscle, the demand by the cells of the intestinal mucosa exceeds the supply. This in turn predisposes to intestinal villus atrophy.

The present invention thus provides a method for treating catabolic dysfunctions in an animal which comprises administering a therapeutically effective amount of glutamine or functional analogues thereof to the animal.

Provision of exogenous glutamine to a stressed patient may better support the metabolic needs of the small intestine and possibly decrease the rate of systemic protein catabolism. Provision of glutamine in patients with inflammatory bowel disease may also be helpful.

It is believed that the therapeutic effectiveness of glucocorticoids in inflammatory bowel disease is not only related to their anti-inflammatory properties, but also to their role in increasing substrate metabolism in the enterocytes of the intestinal epithelium. Administration of exogenous glutamine can provide even more substrate for the enterocytes and can also prevent glutamine deficiency in plasma and skeletal muscle. Similarly, glutamine may improve the survival of transplanted small intestine or support intestinal metabolism in children with intestinal immaturity.

Figure 1 shows the nitrogen balance deposited as a function of

the nitrogen intake.

Figure 2 graphically shows data generated in Example 5 comparing the total amount of branched chain amino acid (BCAA) in arterial blood with the rate of BCAA administration. Data represent mean ± SEM. Data from animals receiving saline are not included. Figure 3 shows graphical data generated in Example 5, which compares the relationship between BCAA flow (back) and

BCAA infusion.

Figure 4 graphically shows data generated in Example 5 comparing BCAA flow (buttock) 6 hours after surgery and the increase in concentration of BCAA in terms of arterial blood level. Figure 5 shows graphical data generated in Example 5, which compares nitrogen flow (buttock) and BCAA flow (buttock) six (6) hours after surgery. Figure:. 6 graphically shows data generated in Example 5 comparing, nitrogen flow (rear) six (6) hours; after surgery and the change in intracellular

amino acid nitrogen in the muscle measured 24 hours later

..operation.

Figure 7 graphically shows the effect of oral diet on plasma glutamine levels after subtotal small bowel resection. Figure 8 shows graphical data demonstrating the effect of oral diet on distal ileum weight after subtotal small bowel resection. Figure -9 shows graphical data demonstrating the effects of oral diet on muscularis thickness in the distal ileum after subtotal resection of the small intestine. Figure .10 shows graphical data demonstrating the effect of r>.'- y:r- •'■ oral diet on mucosal thickness in the distal ileum after

subtotal resection of the small intestine.

Figure 11 graphically shows the effect of oral diet on villus height in the distal ileum after subtotal resection of the small intestine.

The inventors have invented a new method for treating catabolic dysfunctions. The present invention comprises administering to an animal suffering from, or likely to develop, a catabolic dysfunction, a therapeutically effective amount of glutamine or a functional analog thereof. This therapeutically effective amount is greater than that present in a normal diet. The normal dietary intake for humans is approx. 2-4 g/day.

A catabolic dysfunction is a condition that induces a catabolic, biochemical reaction in which a breakdown of an anatomical structure occurs. The dietary administration of glutamine appears to satisfy the biochemical needs of these catabolic states, so that it is not necessary for the body to synthesize glutamine or to obtain glutamine by breakdown of skeletal muscle.

The present invention is intended to be used for all catabolic dysfunctions where there is an increased need for. glutamine. These catabolic dysfunctions can be either enteral or parenteral. The atrophy of villi in the small intestine, which for example occurs when TPN diets are administered, is an enteral, catabolic dysfunction. Villi atrophy does not usually occur due to direct catabolic activity on the enterocytes, but rather is caused by the lack of glutamine in the diet of patients on a parenteral diet.

Parenteral, catabolic dysfunctions showing an increased need for glutamine occur during surgery, sepsis, burns, anorexia and uncontrolled diabetes.

The invention is effective in those animals usually classified as mammals, including humans.

The term "enteral" is meant to indicate the part of the digestive tract that lies between the stomach and the anus.

The term "parenteral" refers to the area outside the alimentary canal.

The term "essentially associated with" when applied to the catabolic dysfunctions for which the method according to the invention is effective, means those where the biological need for glutamine occurs during or after the catabolic dysfunction, and is associated with it.

The administration of glutamine can take place both with enteral and parenteral means.

An example of. how the enteral administration of glutamine is carried out is by using small diameter tubes placed via the nose into the stomach or duodenal areas, or by surgical implantation such as in gastrostomy or jejunostomy.

Examples of parenteral routes of administration include, but are not limited to, such routes as subcutaneous, intramuscular or intravenous injection, nasopharyngeal or mucosal adsorption or transdermal absorption. In most cases, the glutamine is administered intravenously. In the case of intravenous administration, the therapeutically effective amount of glutamine is present in a liquid form which is administered from a reservoir directly via the placement of a needle in a large vein of the patient, in which the needle is connected to the reservoir by means of Tør.

Regardless of which method of administration is used, the glutamine can be administered either alone or as a dietary supplement. When used as a dietary supplement, the glutamine may be mixed with an existing enteral or parenteral diet prior to administration to the patient. It is also possible to administer the glutamine without mixing it directly with other components of a diet as, for example, by intravenous feeding, where the glutamine is not directly added to the main intravenous bottle, but is instead added to a common reservoir using a "ride on your back" bottle.

Functional analogues, derivatives, substitution products, isomers or homologues of glutamine which retain the properties of glutamine are considered to be equivalent. Analogues that can donate an amino group and are metabolized in the Krebs cycle are preferred.

Most preferred are compounds which have the amino acid residue at one end of the carbon chain and an amine part at the other end of the carbon chain.

The therapeutically effective dosage ranges for the administration of glutamine are those large enough to prevent catabolism or atrophy of the body tissues to maintain metabolic homeostasis. In an enteral diet, glutamine will be administered in an amount greater than or equal to 0.3 g/kg body weight/day. Such administration amounts may be 0.3 to 2.0 g/kg body weight/day, preferably 0.3 to 1.5 g/kg body weight/day, and more preferably 0.4 to 1.0 g/kg body weight/day. The administration amount for glutamine when administered intravenously will be greater than or equal to 0.1 g/kg body weight/day. Such administration amounts can be 0.2 to 3.0 g/kg body weight/day, preferably 0.3 to 2.5 g/kg body weight/day, more preferably 0.4 to 2.0 g/kg body weight/day.

In accordance with the method according to the invention, glutamine can be administered by simply modifying existing diet mixtures so that they contain the correct glutamine concentration. Most preferably, the glutamine will remain in a dry form such as e.g. a sterile, lyophilized powder that is aseptically hydrated at the time of administration and mixed in the correct concentration with the other components of the dietary mixture. Alternatively, the glutamine can be premixed with the other ingredients in a dry mixture that is rehydrated aseptically at the time of administration, or stored as a frozen concentrate that is thawed and mixed to the correct concentration at the time of use.

The use of glutamine by means of the method according to the invention is ideally suited for the preparation of mixtures. These mixtures may include glutamine alone or in combination with other chemicals. These other chemicals can be pharmaceutically acceptable carriers, as well as other active substances in the diet such as e.g. free amino acids, protein hydrolysates or oils.

Preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions and emulsions. Carriers or absorbent dressings can be used to increase skin permeability and increase absorption.

The invention also applies to a drug or a pharmaceutical preparation comprising the components according to the invention, as the drug is used for inhibiting catabolic dysfunctions.

Invent late. glutamine-rich mixtures for preventing or improving catabolic dysfunctions also apply. Formulas that are glutamine-rich contain glutamine in amounts that are therapeutically effective and are greater than those present in a normal diet.

Containers containing the composition according to the invention can be used to facilitate the administration of glutamine in accordance with the method according to the invention. These containers are, for example, intended to contain the daily dosage of glutamine to be administered to the patient. Containers adapted for intravenous administration of glutamine alone or in combination with other amino acids are particularly useful. Such containers may comprise a container for the liquid glutamine-containing mixture and a fluid-conducting device that can be attached to a needle. The liquid-conducting device can be any object that can transfer the liquid mixture in the container to the needle, such as e.g. a plastic tube.

The needle attached to the conducting device can either be inserted directly into a blood vessel of the patient or can be inserted into a container to allow mixing with another solution before it is administered to the patient.

The above description describes the invention in general. A more complete understanding may be obtained by reference to the following specific examples which are provided herein for illustrative purposes only and are not intended to be limiting unless otherwise noted.

Example 1

Animal care and operative procedures

22 bastard dogs weighing between 20 and 40 kg were purchased from a farm where they had been trained regularly and where parasites had been removed from them. All female animals were non-pregnant. While in the kennel, the animals were cared for in accordance with the guidelines of the Committee on Animals at Harvard Medical School and the Committee on Care and Use of Laboratory Animals of the Institute for Laboratory Animal Resources, the National Research Council (DHEW Publication #NIH 78- 23, reviewed 1978). The animals were kept in individual kennels at a constant temperature of 20°C with 24 hours of light exposure. They were trained for 2 hours every morning, given water ad libitum, and fed once a day between 1.00 and 3.00 in the afternoon with Agway Respond 2000 Dry Dog Chow® (contains at least 25% protein, 10% fat and the remaining calories as carbohydrate). The dogs were allowed to acclimatize to kennel conditions for 5 to 7 days, during which time they were trained to rest quietly in a Pavlovian position. On the day before the main tests were taken, all food was removed from the kennel at 5.00 in the afternoon. After an overnight fast, the dog was walked for at least 20 minutes, it was placed in a Pavlovian position and the vein on the foreleg was cannulated. After the dog had rested in the position for at least 20 minutes, a venous blood sample was obtained for amino acid determination. After rapid induction of anesthesia with intravenous sodium thiopental (Abbott Laboratories, 5 mg/kg body weight), a biopsy of the vastus lateralis muscle was performed using the method of Bergstrøm et al., Journal of Applied Physiology, 36; 693-697 (*1974). The animal was then removed from the position and a 5 ml sample of arterial blood was obtained from the femoral artery via percutaneous puncture.

The animal was allowed to recover from the biopsy for at least 2 days before the standard surgery. The day before the operation, all food was again removed from the kennel at 5.00 in the afternoon. At At 7.00 in the morning the dog was allowed to walk for 20 minutes and was then taken to the operating theater where it was anesthetized with intravenous sodium pentobarbital (Abbott Laboratories, 30 mg/kg body weight). An endotracheal tube was placed and the animal was allowed to spontaneously breathe a mixture of oxygen and room air. The dog was placed on an operating table supine and a cannula was placed by percutaneous puncture in the external jugular vein and directed into the superior vena cava. After noting the start time, the infusion solution was administered via this cannula by constant infusion (IMED pump®, San Diego, CA) at 4 ml/hour/kg. Penicillin G (E.R. Squibb, Princeton, NJ, 600 mg) and Keflln® (Eli Lilly, Indianapolis, IN, 1 g) were given intravenously. The bladder was catheterized, the first urine sample was discarded, and the catheter was connected to a closed urine bag for collection for 24 hours. The abdomen and flanks of the dog were shaved and the skin was washed with soap and water and prepared with a povidone-iodine preparation solution (Clinipad Corporation, Guilford, CT). The dog was draped with sterile sheets and the abdominal cavity was entered via a vertical infra-umbilical incision in females and a right paramedial incision in males. The intestines were pulled into the upper abdominal cavity and an incision was made in the exposed retroperitoneum. The right artery and vein iliacea circumflexa profunda and artery sacralis media were isolated by sharp and blunt dissection. A specially prepared catheter consisting of a 6 cm segment of polyethylene tubing (2.08 mm OD) coated with silastic and tied to a 2.8 mm OD polyethylene catheter was inserted 6 cm cranially into the aorta via a right iliac circumflexa profunda artery. A similar catheter was inserted into the media sacral artery, its tip being placed approximately 1 cm proximal to the bifurcation of the aorta, but distal to the inferior mesenteric artery. A third catheter was introduced into the inferior vena cava via the right vena iliace circumflexa profunda and placed distal to the renal vein. All catheters were secured and led to the exterior through puncture wounds in the side. The abdominal cavity was then closed and the animal turned on its left side. The external catheters were cut to appropriate lengths, plugged with blunt needles connected to intermittent injection ports (Jelco®, Citikon, Inc., Tampa, FL), flushed with saline, filled with heparin (1000 units/ml), and buried subcutaneously. The injection ports were placed high on the animal's side under the skin and in approximate proximity to the spine. This enabled access to the aorta and vena cava by percutaneous puncture of the injection ports of the catheters. Two further doses of Keflin® (1 g) were given 8 and 24 hours after the operation via the venous catheter.

After the surgical procedure, the animal was placed on its side and body temperature was maintained with heat lamps and blankets during recovery of consciousness. Approximately 5 hours after the start of the infusion, the animal was placed in a pavlov position and a solution of para-aminohippuric acid (PAH, 0.5$, w/v in saline) was infused at a rate of 0.76 ml/min with a Harvard pump in the distal aorta through the catheter in the aorta sacralis media. After 40 minutes of dye infusion, arterial and venous samples were simultaneously obtained for measurement of amino acid and PAH concentrations. Three sets of samples were drawn at 10 minute intervals for a period of 20 minutes. The catheters were then cleaned, filled with heparin and the animal was held in a pavlov bandage. 23 hours after the start of the experiment, the hindlimb flow examinations were repeated. After 24 hours, the urine collection was finished. The animal received intra-venous sodium thiopental, as previously described, and a biopsy was performed and the vastus lateralis muscle in the leg that had not previously been biopsied. The intravenous infusion was terminated and the animal placed in a metabolic cage for the following 24 hours, where water was given ad llbltum and nothing else.

Example 2

lnfus. 1 solutions

All animals received an infusion at a rate of 4 ml/hr/kg. Five control animals received 0.9$ saline solution. Other animals were given commercially available amino acid solution (FreAmine III®, American McGaw) at two different concentrations calculated to deliver approximately 0.312 (N=2) or 0.624 (N=6) g nitrogen/24 h/kg body weight. The highest dose was calculated to provide the equivalent of 4 g protein/24 hours/kg body weight. Three animals received a solution containing glutamine corresponding to 0.312 g nitrogen/24 hours/kg. Finally, one group (N=6) received a mixture of equal amounts of glutamine and FreAmine®, providing nitrogen in an amount of 0.624 g/24 hours/kg. The glutamine solutions were prepared by dissolving L-glutamine (Sigma, St. Louis, MO) in distilled water to form a 0.157 M solution which was then adjusted to pH 6.8 with sodium hydroxide. This solution was sterilized by filtration through a 0.22 µm membrane and stored at 4°C for less than 24 hours. On the morning of use, the solutions were mixed at desired concentrations in 2-liter bags (American McGaw) and kept at 4°C until use. A 10 ml sample was taken from each bag at the end of the infusion and stored at -20°C for analysis of nitrogen content. An additional 10 mL sample was adjusted to pH 4.75 as described below and stored frozen for analysis of glutamine content.

Example 3

Preparation and analysis of samples

Samples of whole blood and plasma were deproteinized by combining with equal volumes of ice-cold, 10% (w/v) perchloric acid and then centrifuged at 3000 rpm. at 4°C for 20 minutes. A 2 ml aliquot of the supernatant was buffered with 0.2 ml of 0.2 M sodium acetate buffer (pH 4.90), adjusted to pH 4.75-4.90 with 5N potassium hydroxide and brought to a final volume of 4 ml with distilled water. The samples were stored at -20°C for later batch analysis of glutamine and glutamate concentrations, using "an enzymatic, microfluorometric assay modified from the method of Lund, "L-glutamine Determination with Glutaminase and Glutamate Dehydrogenase", in Methods of Enzymatic Analysis, vol. 4, Bergmeyer (publisher), Academic Press, New York, 1974, pp. 1719-1722.

During the muscle biopsy procedure, a stopwatch was started immediately when the tissue was removed. The muscle was dissected free of fat and connective tissue and divided into two different parts. Both samples were weighed at least four times during the following two minutes and the weight and time after biopsy were recorded. Current wet muscle weight at time zero was calculated from the best-fitting linear regression of weight deposited against time. The smallest sample (about 15 mg) was dried to a constant weight in a 90°C oven, and the weight of dry, fat-free solids was obtained after extraction in petroleum ether. This sample was then digested in 250 µl of 1N nitric acid and the chloride content was measured by titration with silver nitrate using a semi-automatic titrator (Radiometer, Copenhagen). Plasma chloride was also determined and intra- and extracellular water calculated using the method of Bergstrøm et al., see above. The second muscle sample (approximately 100 mg) was homogenized in 0.5 ml of ice-cold perchloric acid (10$, w/v) with a Polytron Homogenizer (Brinkman, Westbury, NY). The homogenate was centrifuged and the supernatant prepared for enzymatic glutamine and glutamate analysis.

At the start of this study, plasma and intracellular glutamine and glutamate concentrations were determined by an enzymatic method previously described (Muhlbacher et al., American Journal of Physiology, 247: E75-E83 (1984)). Concentrations of other amino acids were determined by automated high-performance liquid chromatography (HPLC) after pre-column derivatization with o-phthalaldehyde. All amino acids commonly found in proteins were quantified except for glutamine, glutamate, proline, cysteine and lysine. As research continued, techniques were developed for glutamine-glutamate measurement using HPLC. Samples measured by the two techniques (enzymatic and HPLC) gave comparable glutamine-glutamate concentrations. Therefore, only HPLC analysis was used in the last part of the investigation. The concentration of PAH in the arterial and venous samples was determined spectrophotometrically after deproteinization with 5% lg trichloroacetic acid (Muhlbacher et al., see above).

Urine excreted during the 24 hours of infusion was collected in the closed urine collection system and stored in acidified, refrigerated containers. Aliquots were stored frozen at -20°C for batch analysis. The nitrogen content of the infusion solution and urine was determined at the same rate using the macro-Kjeldahl method (Peters et al., Quantitative Clinical Chemistry, vol. II, William & Wilkins, Baltimore, MD, 1932, pp. 516-538) . ,

Statistical calculations were performed on an IBM 4341 computer using a standard statistical package (Minitab, The Pennsylvania State University, State College, PA, 1983). The results are expressed as mean ± SEM. Paired and unpaired Student's t-tests were used as appropriate. Analysis of variance was used for multiple group comparisons. Regression analysis was performed using the methods of least squares. Because of the small sample size in the groups receiving 0.312 g nitrogen/24 h/kg, most statistical comparisons were only performed between the other groups.

Buttock blood flow was calculated as previously described (Muhlbacher et al., see above), and velocity was expressed per kg body weight to take into account variations in the size of the animals. Amino acid flow rates were calculated as the product of blood flow and differences in arterial and venous concentration. Three sets of samples were taken, flow was calculated for each set and the mean of the three values determined (Muhlbacher et al., see above). Total amino acid nitrogen in whole blood, plasma and intracellular water was calculated by taking into account the nitrogen content of each amino acid and summing up the individual concentrations.

Example 4

Plasma and intracellular amlnoic acid concentrations. 1 oner

Plasma amino acid concentrations were measured pre-operatively and 24 hours after the standard operation. In the salt-treated animals, the total nitrogen content in plasma was unchanged by the operative procedure (Table 1). The glutamine concentration remained constant, but the branched-chain amino acids increased, the sum of their concentrations increasing from 326 ± 21 to 501 ± 9 pmol/1 (p < 0.01). In the animals that received 0.624 g N/24 hours/kg, there was an upward trend in the plasma nitrogen concentration that was statistically significant only in the group that received the mixture of amino acids plus glutamine. The plasma glutamine concentration also increased in this group. Branched-chain amino acids were increased in all animals receiving amino acid infusions.

The concentration of nitrogen in the skeletal muscle decreased during saline infusion (Table II). This decrease in total amino acid nitrogen was reflected primarily by a drop in glutamine from 21.48 ± 3.21 jjmol/1 intracellular water to 15.86 ± 3.80 (p < 0.05). Although the sum of the concentrations of non-essential amino acids decreased, the sum of total essential amino acids in the intracellular store remained unchanged. No change in intracellular nitrogen or glutamine occurred in animals receiving 0.624 g amino acid nitrogen/24 hours/kg (Table II). There was an upward trend in the intracellular concentration of branched chain amino acids upon infusion of the higher amino acid charges, although statistical significance was only achieved in those animals receiving the mixture of amino acids and glutamine. There was no significant change in the total concentrations of essential and non-essential amino acids in these two groups after surgery. In contrast to the animals receiving the highest dose of nitrogen, the five animals infused with 0.312 g N/24 hr/kg did not maintain the intracellular skeletal muscle nitrogen store consistently, regardless of the infused solution. Intracellular glutamine decreased in three of the animals, remained unchanged in one, and increased in one (data not shown).

By supplying an amino acid equivalent to 0.624 g N/24 hours/kg as an amino acid mixture with or without glutamine, the intracellular skeletal muscle amino acid store was thus maintained. A decrease in the intracellular store, characterized by a drop in intracellular glutamine, occurred consistently in the animals receiving saline and was variable in the animals receiving the lower dose of amino acids. '•"

Net hindlimb amino acid flow, calculated as the sum of the nitrogen flow of the individual amino acids, averaged 19.05 ± 4.06 pmol N/min/kg when measured 6 hours after surgery in the saline animals. This was significantly greater than the efflux rates of -7.70 ± 5.9 and -6.50 ± 1.18 pmol N/min/kg observed in the groups of animals receiving 0.624 g amino acids/24 h/kg ( However, glutamate efflux from the hindquarters was unchanged within these three groups. In contrast, branched-chain amino acids were released in the saline-only dogs but occupied in both groups of animals receiving the higher dose of amino acids. The hindquarter exchange of branched-chain amino acids appeared to be associated with the rate of branched-chain amino acid administration The demonstrated release of branched-chain amino acids from the hindquarters in the saline-treated group balances with the solution containing amino acids plus glutamine and greater uptake in the group receiving the highest dose of branched-chain amino acid. In the five animals that received 0.312 g N/24 hours/kg, there was no significant change in hindquarter nitrogen outflow compared to the saline-treated dogs. However, there was considerable variation in these flow data and the number of animals examined was small. Investigations of hindlimb amino acid flow 24 hours after surgery showed no differences between groups (Table

III).

Nitrogen excretion in the five animals infused with saline was 0.492 ± 0.022 g N/24 hours/kg. In the six animals receiving the highest dose of commercial amino acid mixture, measured nitrogen intake was 0.632 ± 0.001 g N/24 hours/kg and nitrogen excretion averaged 0.684 0.031 (Table IV). In the six animals that received the solution made from one-half commercial amino acid solution and one-half glutamine, nitrogen intake was comparable, but excretion was greater, averaging 0.775 ± 0.019 g N/24 h/kg (p < 0.05 ). The nitrogen balance in these two groups was significantly less negative than in the animals that received saline solution, and amounted to an average of -0.052 0.031 and - 0.140 ± 0.022 g N/24 hours/kg, respectively. In the five animals receiving approximately 0.312 N/24 hr/kg, the mean nitrogen excretion was midway between that observed in the saline controls and in the animals receiving the largest amount of infused nitrogen. Overall, these studies demonstrated that nitrogen balance approached equilibrium as the amount of administered nitrogen increased (Figure 1). When glutamine was combined with a commercial, glutamine-free amino acid solution, the effects on nitrogen balance were additive. When summed together, the nitrogen retained in response to the infusion of commercial amino acids or glutamine alone equaled the nitrogen retained when the solutions were combined.

These investigations show that operative stress in dogs stimulates net protein breakdown in the skeletal muscle, as evidenced by the negative nitrogen balance and increased amino acid outflow from the hindquarters in connection with a drop in the free amino acid store in the Intracellular skeletal muscle. Previous studies have shown that protein loss is not associated with fasting or anesthesia, but is clearly a response to operative stress (Kapadia et al., Surgical Forum, 33: 19-21 (1982)). The release of amino acids from the hindquarters 6 hours after surgery in the saline-treated group was approximately 6- to 8-fold greater than that observed in chronically catheterized, post-absorptive dogs examined under basal conditions (Muhlbacher et al., American Journal of Physiology, 247: E75-E83 (1984)). This rate of nitrogen release from the hindquarters cannot be explained by depletion of the intracellular free amino acid store and must therefore reflect a net skeletal muscle proteolysis.

The supply of amino acids in the perioperative period balances the nitrogen loss, retained or increased, plasma amino acid concentration erosions and reduces the drop in the intracellular, free amino acid store in the skeletal muscle. These effects appear to be related to the amount of amino acid nitrogen infused. Nitrogen loss in the whole body and in the hindquarters was greatly reduced at the highest amino acid doses, which also maintained intracellular levels of glutamine and other amino acids. These results differ from the findings reported by Askanazi et al, Annals of Surgery. 191: 465 (1980), who described a decrease in the intracellular concentrations of glutamine and other amino acids in patients after hip replacement that could not be reversed by infusion of dextrose and amino acids. Results obtained using the method according to the invention indicate that these previous findings may be related to the amount of infused amino acids and/or the lack of glutamine in the infusate. Infusion of lower concentrations of amino acids (0.312 g N/24 h/kg), either as glutamine alone or as FreAmine®, failed to maintain intracellular amino acid stores in three of the five animals examined. In contrast, the higher rate of amino acid infusion stabilized or increased the intracellular store. It thus appears that an adequate amount of administered nitrogen can maintain the intracellular amino acid store in the skeletal muscle post-operatively.

The change in the intracellular free amino acid store in saline-infused animals, which is largely attributable to a rapid drop in glutamine, was prevented when adequate nitrogen was supplied. This occurred even when glutamine was not present in the commercially available solution. The mechanism by which intracellular glutamine was maintained under these circumstances is unclear, although it seems likely that glutamine substrate for glutamine synthesis was obtained from the branched-chain amino acids via transamination. For unexplained reasons, net glutamine efflux was similar in all groups. Hind end release of glutamine was not accelerated by branched-chain amino acids or decreased by addition of glutamine in the amino acid solution. The results in this post-operative model differ from reported effects of branched-chain amino acids in normal humans, in which forearm uptake of branched-chain amino acids after oral administration of leucine was associated with accelerated glutamine release see (Aoki et al., Journal of Clinical Investigation, 65: 1522 (1981)).

Even there were marked differences in the composition of the two amino acid solutions administered in; an amount of 0.624 g N/24 h/kg, hindquarter nitrogen efflux was comparable in both groups of animals. This occurred even though the amount of essential amino acids and branched-chain amino acids in the balanced solution was twice as great as in the glutamine-containing solution. Thus, in this experimental model for operative stress, glutamine supplementation of a balanced amino acid mixture was at least as effective as twice the concentration of the standard balanced mixture in reducing hindlimb loss.

In the dogs that received the saline solution, branched-chain amino acids were released from skeletal muscle. Quantitative transfer amounts calculated from these data suggest that there must have been a marked uptake of branched-chain amino acids into visceral organs, most likely the liver, during the earlier post-operative period. The supply of branched-chain amino acids appears to offset this translocation. , perhaps by both satisfying vascular demands and reversing skeletal muscle efflux. A quantitative relationship also exists between hindlimb nitrogen balance and protection of the intracellular nitrogen store. When intracellular stores were maintained, there was nearly nitrogen equilibrium in the hindlimb. When saline was administered, amino acid concentrations in the intracellular store was markedly depleted and there was a marked loss of hindlimb nitrogen.Although the relationship between skeletal muscle proteolysis and nitrogen concentration in the free amino acid store is unknown, these data suggest that skeletal muscle nitrogen balance is related to intracellular amino acid concentration and that gluta mine can play a decisive role in maintaining a riohomeostatic balance in the body.

Example 5

Branched-chain amino acid uptake and free amino acid concentrations in the muscle predict post-operative nitrogen balance in the muscle.

To investigate the effectiveness of BCAA infusion in reducing protein catabolism in skeletal muscle and the whole body, amino acid mixtures containing different concentrations of BCAA were given perioperatively in this study to three. groups of dogs subjected to a standard laparotomy and retroperitoneal dissection. A fourth group was given saline only. Using posterior flow techniques, individual and total amino acid-nitrogen exchange rates were measured and used in the determination of protein catabolism in skeletal muscle. Intracellular free amino acid concentrations were measured in percutaneous muscle biopsy specimens. The work focuses on the effects of intra-nasal amino acid solutions containing different concentrations of BCAA and the amino acid metabolism in skeletal muscle after a standardized surgical procedure on the dog. By measuring hindlimb amino acid flow and free amino acid concentrations in plasma and skeletal muscle during the first 24 hours after surgery, it has been possible to evaluate the anti-catabolic response to the infusion of BCAA and other amino acids.

Materials and methods

Animal preparation and examination sequence

27 male and non-pregnant female bastard dogs were purchased from a farm where they were trained and kept free of parasites. The dogs weighed between 18 and 40 kg and were housed for at least one week prior to the study in the Harvard Medical School Animal Care Facility. All procedures were carried out in accordance with the guidelines of the Committee on Animals at Harvard Medical School and the Committee on Care and Use of Laboratory Animals at the Institute for Laboratory Animal Resources, National Research Council, see above. The animals were housed individually with 24 hours of light exposure and were exercised every morning. Water was provided ad libitum, and a single daily feeding of Pro-Pet Respond 2000 dry dog food (Syracuse, New York, at least 25 wt-$ protein) was given between 1.00 and 3.00 in the afternoon. The animals were trained to rest quietly in a Pavlov loop before the examination.

Everything was removed from the kennel at 5.00 in the afternoon before the basal examinations or surgery. The basal examinations were carried out at 8.00 in the morning after the animal had been exercised and placed in the sling. These examinations consisted of collecting one blood sample from a cannulated foreleg vein for plasma amino acid determination and a percutaneous needle biopsy of the vastus lateralis muscle was performed under sodium thiopental anesthesia (Abbott, North Chicago, Illinois, 5 mg/kg body weight, IV) for to determine intracellular free amino acids. After the biopsy, while the dog was still anesthetized, a 5 ml sample of arterial blood was obtained by percutaneous puncture of the femoral artery for analysis of amino acids in whole blood.

The animal was allowed to recover for 3 days before further investigations were carried out. At 7:00 AM on the day of surgery, again after an overnight fast, the animal was exercised and taken to the operating room where it was anesthetized with sodium pento-barbltal (Abbott, North Chicago, Illinois, 30 mg/kg body weight, IV) via a foreleg cannula. An endotracheal tube was placed and the animal was allowed to breathe spontaneously a mixture of room air and oxygen supplied at a rate of 5 l/min. The dog was placed on an operating table lying on its back and a 16-Fr. catheter was placed percutaneously in the superior vena cava via the external jugular vein. After noting the start time, infusion of either saline or the appropriate test amino acid solution was begun via this central catheter with IMED pump (San Diego, California). Cephalothin (Lilly, Indianapolis, Indiana, 1 g, IV) was given immediately before and after completion of surgery. The urinary bladder was catheterized, and after discarding residual urine, a closed drainage collection was started at the start of the infusion and continued for 24 hours. Urine was also collected for a second 24 hour period, with the animal in a metabolic cage or termination of the IV infusion.

The dog's belly and sides were shaved, washed with soap and water and prepared with a povidone-iodine solution. The animal was draped sterile and the abdomen was entered via an infra-umbilical midline incision in bitches and a right paramedian incision in male dogs. The viscera were pulled aside and the retroperitoneum exposed for complete dissection around the distal aorta and inferior vena cava. The right artery and vein iliacea circumflexa profunda as well as the right internal artery iliacea were isolated. The two arteries were cannulated with specially prepared catheters consisting of a 6 cm segment of polyethylene tubing (2.08 mm OD) connected to a 2.8 mm OD polyethylene tubing. An arterial catheter was placed 6 cm proximally into the aorta via the circumflex iliac artery and the other catheter was placed 1 cm proximal to the aortic bifurcation, but distal to the mesenteric caudale artery, via the interna iliac artery. A third catheter was introduced into the inferior vena cava via vena iliace circumflexa profunda and placed distal to the renal vein. All catheters were fixed and brought out through puncture wounds in the right side. The abdomen was closed in layers and the animal turned on its left side. The deployed catheters were divided to appropriate lengths, plugged with blunt needles attached to intermittent injection ports (Jelco, Critikon, Tampa, Florida), flushed with saline, filled with heparin (100 pU/ml), and buried subcutaneously. The injection ports were placed high on the sides, to allow easy access to arterial (aortic) and venous (from vena cava) blood by percutaneous puncture.

After these procedures, which generally took 2 hours, the animal was placed on its side and body temperature was maintained with blankets during recovery from anesthesia. 5 hours after the start of infusion and surgery, the animal was placed in the Pavlov loop and a solution of 0.5% para-amino-hiipurate (PAH) was infused at a rate of 0.7 ml/minute with a Harvard pump 1 the distal aortic catheter. After 40 minutes of dye infusion, three sets of simultaneous arterial and venous samples were obtained at 10-minute intervals for measurement of amino acid and PAH concentrations. The catheters were then separated and filled with heparin. The animal was kept in the sling under constant monitoring until the hindlimb flow examinations were repeated 24 hours after the start of the infusion. At this point, the initial 24-hour urine collection was terminated and a repeat percutaneous hindlimb biopsy was performed on the previously unbiopsied leg, again under brief general anesthesia. The infusion was then terminated and the animal placed in a metabolic cage for the second 24-hour period.

Infusion solutions

All solutions were infused at a rate of 4 ml/min/kg. Five control animals received 0.9$ saline solution. Amino acid solutions (Table V) containing BCAA 1 at three different concentrations (11%, 22%, or 44% of total amino acids) were prepared by adding amino acids to an 8.5% standard amino acid mixture, FreAmine III (Amercian McGaw, Irvine, California). . They. total BCAA infusion rates were 0.46, 0.92 and 1.84 g/24 hours/kg, respectively. All three amino acid solutions were isonitrogenous, yielding approximately 0.624 g nitrogen/24 h/kg, with a constant ratio of valine to leucine to isoleucine (1:1.38:1.05). Nine animals received an 11$ BCAA solution prepared by dissolving a mixture of non-essential amino acids (NEAA) in 2.13$ FreAmine III to obtain. a solution that gave 0.624 g nitrogen/24 hours/kg. In 6 animals, NEAA consisted of L-glutamine alone and 1 three, NEAA consisted of a mixture of all the NEAAs found in FreAmine III (alanln, glycln, arginine, histidine, serine and proline) in the same ratio as 1. FreAmine III. Six animals received 4.25$ FreAmine III alone (22$ BCAA). The last 7 animals received 2.13$ of FreAmine III with enough BCAA added to form a 44$ solution. This latter mixture was made isonitrogenous by adding NEAA as L-glutamln alone (n = 4) or a mixture of NEAA contained in FreAmine III (n = 3). All solutions were sterilized by passage through a 0.22 µM filter (Millipore, Millis, MA) and stored overnight at 4°C before administration. A 10 ml sample of each solution was taken at the end of the infusion period and stored at -20°C for analysis of nitrogen by the macro-Kj eldahl method.

Preparation and analysis of blood, tissue and urine samples

Samples of whole blood and plasma were deproteinized by adding an equal volume of ice-cold 10 µg perchloric acid (PCA) and then centrifuging at 7000 rpm. at 4°C for 20 minutes. A 2 ml aliquot of the supernatant was buffered with 0.3 ml of 0.2M sodium acetate buffer (pH = 4.90), adjusted to pH 4.75-4.90 with 5N potassium hydroxide, brought to a final volume of 4 ml with distilled water and centrifuged again. The resulting supernatant was stored at -20°C for later batch analysis.

During the muscle biopsy procedure, a stopwatch was started at the time of tissue removal. The muscle was dissected free of fat and connective tissue and divided into two different parts. Multiple weights of each sample were recorded at 15 second intervals for one minute, and the initial wet muscle weight at time = 0 was calculated from the best fit linear regression of weight deposited against time. The smaller sample (about 15-20 mg) was dried to a constant weight in an oven at 90°C and the weight of dried, fat-free solids was obtained after extraction with petroleum ether. The sample was then immersed in 250 ml IN nitric acid and the chloride content was measured by titration with silver nitrate using a semi-automatic titrator (Radiometer, Copenhagen). Plasma chloride was also determined using a similar method. Intracellular and extracellular water was then calculated using the chloride technique, as described previously. The second muscle sample (approximately 80-100 mg) was weighed and homogenized in 0.5 ml of ice-cold PCA using a Polytron homogenizer (Brinkmann, Westbury, New York). The homogenate was centrifuged and the supernatant was prepared for analysis by adding buffer and adjusting the pH to pH 4.75-4.90 as described for blood and plasma samples. Intracellular glutamine and glutamate concentrations in whole blood, plasma and muscle were determined by an zymatl, microfluorometric method modified from the method of Lund, P., "L-glutamine determinatlon with glutaminase and glutamate dehydrogenase," in "Methods of Enzymtic ANalysis" , Bergmeyer, H.U., publishers, vol. 4, New York: Academic Press 1719-1722 (1974), or by automated high-performance liquid chromatography (HPLC) after pre-column derivatization with o-phthalaldehyde, ^derivatization with o-phthalaldehyde, Smith, R.J., et al., " Automated analysis of o-phthaladehyde derivatives of amino acids in physiological fluids by reverse phase high performance liquid chromatography," J. Lig. Chromatog. 8: 1783-1795 (1985). The two techniques produced comparable results. Other α-amino acids except proline, cysteine and lysine were assessed by a similar HPLC method. The concentration of PAH in arterial and venous blood was determined spectrophotometrically after deproteinization with 5% trichloroacetic acid, Muhlbacher, F., -et.al., "Effects of glucocorticolds on glutamle metabolism- in skeletal muscle," Am. J. Physol. 247: E75-E83

(1984).

Urine excreted during the 24 hours of infusion was collected in a closed urinary drainage system and stored in acidified, refrigerated containers. Aliquots were stored frozen at -20°C for later batch analysis of nitrogen by the macro-Kjeldahl method, Peters J.P., et al., "Total and non-protein nitrogen", in Quantitative Clinical Chemistry, vol. II, 516-538, Baltimore: ; Williams & Wi 11 lambs, (1932). Another portion was centrifuged for 10 minutes: at 2000 rpm. and frozen for later analysis of urea and creatinine on the Technicon Auto analyzer (Tarrytown, New York).

Calculations and statistical analysis

Buttock blood flow was calculated as described above (Muhlbacher, F. et al., see above). Flow rates for the individual amino acids were calculated as the product of blood flow and arteriovenous concentration difference. Three sets of samples were taken at each time point, the current was calculated for each set and the mean of the three values was determined. Total amino acid nitrogen flux as well as plasma, whole blood, and intracellular nitrogen concentrations were calculated as the millimolar sum of the nitrogen groups of all measured amino acids. Concentrations of free, intracellular amino acids in the skeletal muscle were expressed per liters of intracellular water.

Statistical calculations were performed using a standard statistical package (Minitab, The Pennsylvania State University, State College, PA, 1983). The results are expressed as the mean ± SEM. Paired and unpaired Student's t-tests were used as appropriate. Analysis of difference when used for comparison of several groups. Regression analysis was performed using the least squares method.

Results

All animals survived the operative procedure except for one dog that died shortly after the administration of sodium pentobarbital, before the start of the intravenous infusion. This animal was not included in the study. Blood loss during the procedure was uniformly minimal. All test catheters were indwelling at the 6- and 24-hour time points, with the exception of a venous catheter at the 24-hour time point in one animal in the 22$ group

BCAAs.

Buttock blood flow at 6 hours was 36.1±6.8 ml/minute/kg in the saline control group and was not affected by treatment (11$ BCAA, 33.3±4.9; 22$ BCAA, 42.4±8, 8; 44$ BCAA, 28.7±3.5-; the differences were not significant). Current at 24 hours was unchanged (respectively 57.9±10.2, 38.6±8.2, 54.9±6.5, 49.7±13.2). The tendency towards higher flow rates and increased variability at 24 hours can be attributed to greater movement activity in the animals after awakening from anaesthesia. Excretion of nitrogen through the urine and nitrogen balance After the operation, the volume of excreted urine was comparable in the four treatment groups, although the dogs that received saline alone tended to excrete a smaller volume of urine (Table VI). The average excretion of nitrogen through the urine was 0.492±0.20 g/24 hours/kg in the saline group. The amino acid-treated animals excreted 35-65$ more nitrogen than the saline group, primarily in the form of urea. The dogs infused with the 22$ BCAA solution excreted significantly less urea nitrogen and less total nitrogen than the 11$ or 44$ BCAA groups. Excretion of creatinine and ammonia was comparable in all groups.

Blood urea nitrogen and plasma creatinine were measured before and 24 hours after the operation in selected animals from all groups. These concentrations were normal in all animals before surgery and decreased slightly or did not change after surgery. Thus, the rate of excretion of urea through the urine was equal to the rate of urea production. The higher urea production observed in the animals receiving solutions with 11$ and 44$ BCAA was significantly related (p < 0.05) to the additional nitrogen provided by the addition of BCAA or NEAA to the balanced amino acid mixture.

The nitrogen balance was less negative with amino acid administration. About 50% of the infused amino acid nitrogen was retained. Because the nitrogen intake was the same in all animals receiving amino acids, the changes in nitrogen excretion that have already been discussed were reflected in the nitrogen balance (Table VI). Thus, the animals that received the solution with 22% balanced amino acid achieved significantly greater nitrogen retention than the dogs that received solutions containing 11% or 44% BCAA.

Amino acid concentrations in whole blood

In the animals treated with saline, the amino acid nitrogen in whole blood fell 6 hours after surgery, but returned to normal, preoperative levels after 24 hours (Table VII). This transient hypoaminoacidaemia was largely due to a decrease in the concentration of the non-essential amino acids (glutamine, alanine, arginine, serine and asparagine), although significant decreases in some essential amino acids also occurred (threonine and tyrosine). In contrast, the animals that received amino acid infusions maintained whole blood amino acid nitrogen concentrations 6 hours after surgery. These levels increased above preoperative control levels at 24 hours (p < 0.05).

The concentrations of particular amino acids in the blood of the animals receiving amino acid infusions reflected the composition of the infused solutions. The BCAA concentrations were, for example, related to the degree of BCAA administration both after 6 and 24 hours (figure 2). In general, glutamine concentrations in whole blood at 6 hours were lower than preoperative levels (Table VII). The exception was the group that received glutamine-enriched, 11% BCAA solution, in which the concentration of glutamine in the blood was maintained. In these animals, glutamine comprised more than half of the non-essential nitrogen and accounted for more than 40% of the total amino acids released. After 24 hours, those animals that received glutamine-containing infusions tended to have higher than normal concentrations of glutamine in whole blood.

Intracellular free amino acids 1 skeletal muscle In the animals treated with saline, intracellular free amino acid nitrogen dropped significantly 24 hours after surgery when compared to preoperative levels (Table VIII). This change could be largely attributed ($65) to the marked drop in intracellular glutamine, which comprised a major part of the total store of intracellular free amino acid. In the animals that received amino acid infusions, Intracellular nitrogen was maintained, although intracellular glutamine fell in the animals that received the glutamine-free solution with 11$ BCAA. Intracellular glutamine tended to increase in those animals receiving glutamine-enriched solutions and BCAA concentrations increased in proportion to the degree of BCAA infusion.

Backdel amino acid flow

In the animals treated with saline, there was a net release of amino acid nitrogen from the hindquarters 6 hours after the operation (Table IX). This increased amino acid efflux reflected accelerated release of nearly all measured amino acids, including BCAAs. At this time period, glutamate and aspartate were the only amino acids that maintained balance throughout the hindquarters. At 24 hours after surgery, the rate of release of hindlimb amino acid nitrogen was decreased and, although highly variable, the arteriovenous differences for almost all amino acids were indistinguishable from zero. Glutamate influx continued at this time.

In all groups of animals receiving amino acid infusions, efflux of hindlimb amino acid nitrogen after 6 hours was similar and was significantly less than in animals treated with saline (p < 0.05). Both glutamine and alanine efflux at 6 hours tended to be less in the animals receiving amino acids than in the saline controls. While. BCAA were released at 6 hours in the animals infused with saline, these amino acids were taken up in the dogs that received amino acid infusions. Hind end uptake of BCAA was related to the degree of administration of BCAA (Figure 3) and total BCAA concentrations (Figure 4).

After 24 hours, hindlimb amino acid nitrogen efflux was similar in all amino acid infusion groups and unchanged compared to 6 hours. After 24 hours, hindlimb BCAA exchange was slightly positive, tending to be greater in the 22 and 44 groups. $ BCAA At this time, BCAA uptake was unrelated to blood concentrations and rate of BCAA administration.

Relationship between BCAA infusion. BCAA uptake and amino acid nitrogen release

In those dogs infused with saline, hindquarter BCAA release None after 6 hours was associated with accelerated amino acid efflux. In those animals that received amino acid infusions, hindquarter nitrogen balance correlated with BCAA uptake (Figure 5). Saline controls were not included in this analysis, as they did not receive nitrogen. Inclusion of control animals would have resulted in a regression line with a more positive slope. The correlation was maintained even though the BCAA flow was not included in the summary of hindlimb amino acid-nitrogen flow (p < 0.02, r = 0.49). Nitrogen flow without BCAA flow was thus also related to BCAA uptake. Nitrogen flux did not correlate with total blood BCAA concentration or rate of BCAA administration. None of these conditions existed at the 24-hour time point.

Release of tail amino acid nitrogen after 6 h also correlated with changes in the intracellular store of free amino acid nitrogen (Figure 6). Changes in intracellular glutamine were closely related to changes in the total store of free amino acid-nitrogen (p < 0.001, r = 0.90) and thus changes in the glutamine store were also significantly related to the hindquarters nitrogen outflow (p < 0.05, r = 0 ,66). These mathematical relationships were maintained when the efflux of tail amino acid nitrogen was corrected for changes in the intracellular nitrogen store. Since the flux and storage measurements were performed at different time points, this correction was made by assuming two different rates of change of the intracellular amino acid storage. First, it was assumed that the change of the intracellular store appeared in the first

6 hours after the operation. Alternatively, it was assumed that

the change occurred at a constant rate during 24 hours. None of the corrections changed the relationship between the change in the intracellular nitrogen store and the amino acid nitrogen efflux.

BCAA uptake was not related to increased glutamine release from the hindquarters after 6 or 24 hours. The BCAA uptake was also not related to the changes that occurred in the intracellular store of free amino acid in the skeletal muscle (r = 0.11, not significant). Hind end nitrogen flow could be predicted and most of the variation in the data could be accounted for when both the flow of BCAA after 6 hours and the change in the store of free amino acid nitrogen were used. The ratio was:

where,

y = amino acid-nitrogen flow after 6 hours, pmol/min/kg, Xj[= BCAA-st escape after 6 hours (jjmol/min/kg),

X2= change in intracellular, free amino acid nitrogen i

skeletal muscle (postop - preop, mmmol/1/24 hours),

n = 22, p < 0.05, r = 0.86.

Discussion on

A standardized laparotomy of anesthetized dogs has been shown to initiate many of the catabolic responses observed in critically ill humans. Total body protein catabolism, as measured by urinary nitrogen excretion, is increased. The control animals that received saline excreted about 12-15 g of nitrogen in the first 24 hours after the operation. Previous investigations in this model have shown that nitrogen balance remains negative for 3 days after the surgical procedure despite nutritional intake. Kapadia, C.R., et al., "Alterations in glutamine metabolism in response to operative stress and food deprivation", Surg. Forum 33: 19-21 (1982). In contrast, pair-fed skin-operated animals achieved nitrogen equilibrium during the first day after surgery. Consistent with and contributing to the increased urinary nitrogen loss, hindlimb release of total amino acid nitrogen 6 hours after surgery was 6 to 8 times greater than that observed in control animals after an overnight fast (Muhlbacher, F., et al., supra ). Other changes in dogs that were treated with saline, such as a decrease in amino acid concentrations in blood and skeletal muscle is similar to the changes reported under catabolic conditions in humans, Askanazi J., et al., "Muscle and plasma amino acids following injury: influence of intercurrent infection", Ann. Sorrow. 192: 78-85

(1980). Thus, the dog model exhibits postoperative responses that are similar to changes in critically ill humans and is therefore suitable for investigating the effects of exogenous amino acids on nitrogen metabolism and amino acid exchange in skeletal muscle.

In the animals that had been treated with saline solution, hindquarter release of amino acid nitrogen was markedly increased 6 hours after the operation. This was associated with a net skeletal muscle release of all BCAAs. At the same time, BCAA concentrations in whole blood were unchanged, indicating that the consumption of BCAA in the visceral organs was roughly equivalent to the accelerated rate of skeletal muscle release. In the animals that received amino acids, total hindlimb amino acid release was reduced, nitrogen stores in whole blood and skeletal muscle were maintained, the hindlimb was converted from an organ with BCAA release to an organ with uptake. BCAA, and more specifically, leucine uptake was not associated with increased skeletal muscle release of glutamine.

This investigation shows that skeletal muscle amino acid release and therefore the net turnover of muscle protein can be predicted from two independent measurements. These are the rates of BCAA flow through the vascular layer. in the skeletal muscle and the concentration of nitrogen in the skeletal muscle's free amino acid stores. Since glutamine is the most abundant amino acid in the free store, changes in its concentration largely determine changes in the total store. Changes in free glutamine actually predict muscle nitrogen balance as well as changes in total free amino acids.

Example 6

Effects of the glutamine drug. oral diet on the small intestine after bowel resection

Introduction

Compensatory growth of the small intestine after partial resection includes all linings of the intestinal wall, but is dominated by villus hyperplasia. Increased villus height and crypt depth is followed by expansion and lengthening of the remaining intestine, Williamson, R.C.N., "Intestinal adaptation", N. Engl. J. Med., 298:1393-1444 (1978). Resection of the small intestine is followed by adaptive, morphological and functional changes. Oral intake has been shown to be an important stimulus in the regulation of mucosal hyperplasia after intestinal resection, Levine, G.M. , et al., "Small-howel resection, oral intake is the stimulus for hyperplasia", Dig. Haze. 21:542-545 (1976). "Luminal" nutrients are important in maintaining normal mucosal growth and if oral intake is continued after resection of the small intestine, the remaining intestine loses weight and becomes hypoplastic. Patients with a short bowel after small bowel resection are supported by parenteral nutrition. Their survival depends on the capacity of the remaining intestinal system to adapt. The use of elemental diets and parenteral nutrition allows sufficient time for the development of intestinal adaptation and a slow return to complete oral intake, Weser, E., "The management of patients after small bowel resection", Gastroenterology, 71:146-150 (1976) .

Glutamine is an important fuel for enterocytes and utilization of it in the gut appears to increase after surgical stress, Souba, W.W. , et al., "Postoperative alteration of arterial venous exchange of amino acids across the gastrointestinal tract", Surgery 94,(2): 342 (1983). Glutamine can serve as a local tropic factor, promoting mucosal growth when intestinal hypoplasia develops. This trial investigates whether the addition of glutamine to an elemental diet was associated with any benefit in adaptation and healing of the small intestine after subtotal resection. The experiment took place by feeding rats a glutamine-enriched elementary diet after 2/3 resection of the small intestine. The mucosal adaptation of the remaining intestine was compared with control diets and sham-operated groups.

Materials and methods

Male Wistar rats weighing 175-200 g were purchased from Charles River Breeding Laboratories, Inc., which were given 5 days to acclimate to observation. The rats had free access to water and were fed a Purina porridge diet. They were kept in individual cages and weighed every other day. After acclimatization, rats with normal weight gain were divided into four groups.

On the first day of the experiment, the rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). The abdomen was opened via a midline incision, the entire length of the small intestine from the tendon of Treitz to the ileocaeal valve was retracted from the abdomen and measured twice without stretching with a long black thread. The mean of the two measurements was determined. A 2/3 resection of the small intestine was then performed beginning 5 cm distal to the Treitz tendon using the method ti Lambert, R., "Surgery of the digestive system of the rats", Charles C. Thomas, Springfield, Illinois , pp. 32-35, 413-416 (1965). The resection edges were anastomosed end to end with 6-0 prolene. The bowel was reinserted into the abdominal cavity and the abdominal cavity wall was closed with 2-0 prolen. A control group underwent skin surgery by undergoing transection and reanastomosis of the small intestine at the distal end, where 2/3 of the total length was measured starting 5 cm distal to the Treitz tendon. The rats were kept in 1 individual cages after the operation and were allowed to drink water on the first day after the operation.

Oral feeding was started from the second day after the operation. Rats 1 group I were orally fed glutamine-enriched elementary diet ($4.18) and rats in group II were fed glycine-enriched elementary diet ($4.18). Rats in group III (resected rats) and rats in group IV (skin-operated rats) were fed with ordinary porridge diet. The feeding was continued for 17 days and their daily body weights were recorded until the last day of the experiment.

Production of glutamine and glysln elementary deletions

The elemental diet (NBCq Biochemicals, Inc., Cleveland, Ohio) contains 0.2$ choline chloride, 10$ corn oil, 46.9$ dextrin white, 23.4$ sucrose, 5$ salt mix, 0.5$ vitamin mix, and 9.82 $ of 17 kinds of essential and non-essential amino acids. Glutamine or glycine powder was added to the elemental diet to produce 41.8$ of glutamine- or glycine-enriched elemental diet, i.e. 28$ of the total amino acids.

Harvesting procedures

After 7 days of feeding, the rats were killed. They were anesthetized with intraperitoneal injection of pentobarbital (50 mg/kg). The abdomen was opened and the incision extended to the chest cavity. 5 ml of blood was withdrawn by puncture of the right ventricle for determination of ammonia and plasma glutamate levels in the blood. The intestine was removed immediately after the blood collection. The entire small intestine was removed with careful separation of the mesentery, keeping close to the serous intestine. The removed intestine was suspended under a fixed tension of 4.5 g and 9 points were marked. For the rats with intestinal resection, points 5, 10, and 12.5 cm proximal to the anastomosis represented the proximal jejunum and distal duodenum, and points 2, 5, and 10 cm distal to the anastomosis represented the remaining proximal ileum near the anastomosis , and points at 5, 10, and 15 cm proximal to the ileocaecal valve that represented the remaining distal ileum, marked with straight pins passed through the intestine. The six intestinal segments were separated. For the rats in the skin-operated group, the proximal two segments were marked from 1 cm distal to the Treitz tendon and measured upwards. Segments 1a, 2a and 3a were rinsed in chilled saline and placed in 10% buffered formalin for 4 hours, then transferred to 70% ethanol for fixation. Segments 1b, 2b and 3b were rinsed in chilled saline, their cavities opened and their wet weight determined. The intestinal segments were transferred to 5 ml of chilled saline and divided with sharp scissors. A homogenate was prepared, using two 15 second periods on a Polytron homogenizer (Brinkman Instruments, Westbury, NY), followed by sonication with an ultrasonicator (Heat system Laboratories, Plannview, NY) at a power of 2, in 30 seconds. The homogenate in salt solution for DNA and protein analysis was stored at -30°C.

Analytical methods

The intestinal homogenates were analyzed for total protein according to the method of Lowry, O.H. et al., "Protein measurement with the Folin phenol reagent", J. Biochem., 193: 265-275 (1951). DNA was determined according to the method of Burton, K., "A study of the conditions and mechanism of the diphenylamine reaction for the c.olorlmetric estimation of deoxyribonucleic acid", Biochem, 6^:315-323 (1965). Histological sections were placed in paraffin, stained with hematoxylin and eosin and examined under a light microscope at 40 times magnification. Twenty representative, large, well-oriented, complete villi were selected to measure mucosal thickness and villus height using an ocular micrometer and an average value obtained. The villus number was counted with the intestine laid in the central horizontal line in a 40x magnified field.

Results

Figure 7 shows that the glutamine-supplemented diet results in higher plasma glutamine levels. Based on weight/cm measurements (Figure 8), there appeared to be little difference between the glutamine-supplemented (+GLN) and glutamine-free diets, both of which supported less intestinal hypertrophy than normal before. However, when examining the intestinal mucosa by measuring mucosal thickness (figure 10) and villus height (figure 11), the glutamine-added diet proved superior.

Example 7

Preservation of small intestinal mucosa using glutamine-enriched parenteral nutrition

Parenteral nutrition results in mucosal atrophy of the small intestine (Johnson, L.R. et al., Gastroenterology 68:1177-1183 (1975)). This response may be related to a decrease in gastrointestinal secretions and trophic hormones and also a relative lack of special nutrients required for enterocyte growth. Glutamine is an important oxidizing fuel for the small intestine, but is not present in standard parenteral solutions. To determine the impact of dietary glutamine, the small intestinal response to the administration of parenteral solutions enriched with varying concentrations of this amino acid was evaluated.

MATERIALS AND METHODS

Conditioned male Wistar rats (n = 42, weight 210-230 g) were subjected to jugular vein catheterization and were fitted with a swivel device allowing long-term infusion in unligated animals (Burt, M.E. et al., J. Physiol., 238:H599 -603 (1980)). All rats were kept in individual metabolic cages and allowed access to drinking water. Control animals received an infusion of 0.9$ saline and Purine rat chow ad libitum. Three groups of rats received intravenous nutrition. All nutrient solutions were isonitrogenous (0.9 g nitrogen/100 ml) and isocaloric (98 Kcal/100 ml), and contained equal concentrations of essential amino acids, non-essential amino acids and dextrose. The non-essential amino acid component of each solution was adjusted to provide glutamine concentrations of 0.2 or 3 g/100 ml. Parenteral solutions were infused at a rate of 48 ml/24 hours. Urine excretion and nitrogen excretion were measured daily. The animals were killed after 7 days of parenteral nutrition and blood was obtained for determination of glutamine and ammonia concentrations. Both full-thickness jejunal segments and mucosal samples were obtained from defined sections of the intestine. All samples were weighed, and homogenates were examined for DNA and protein. Histological paraffin sections of 5 µm thickness were prepared. Measurements of jejunal villus height, number and mucosal thickness were performed in a blind test.

RESULTS AND DISCUSSION

Wet weight, DNA, protein and villus height decreased in all rats receiving intravenous nutrition compared to orally fed controls (Table X). Plasma glutamine concentration increased after infusion of solutions containing glutamine. Jejunal mucosa weight increased significantly compared to two rats receiving glutamine-free solutions. Full-thickness jejunal weight did not tend to increase with glutamine intake, although the response was not statistically significant. DNA both in the mucosa and full thickness of the jejunum increased after glutamine infusion in 2 and 3$ concentrations. These changes were accompanied by histological evidence of mucosal growth. Villus height and mucosal thickness increased in a dose-dependent manner in relation to the amount of glutamine administered. All animals were in positive nitrogen balance, but rats receiving 2$ glutamine solution retained the greatest amount of nitrogen throughout the investigation.

The supply of glutamine in parenteral solutions results in an increase in jejunal mucosa weight, DNA content and villus height when animals are maintained on intravenous nutrition. An increase in the mucosal mass in the small intestine can improve the function of the small intestine and facilitate the introduction of enteral nutrition. Glutamine may be a nutrient that is necessary for the support of the mucous membrane which is not present in standard parenteral solutions at present.

Example 8

Effect of glutamine-enriched. parenteral nutrition after treatment with 5-fluorourlcil

Addition of the amino acid glutamine (GLN) to nutrient solutions significantly increases intestinal cellularity during parenteral feeding. To assess the effect of GLN on mucosal regeneration after injury, 5 fluorouricil (5FU) was administered to rats receiving parenteral nutrition (PN). After jugular vein catheterization, male Wistar rats (n=40, weight 200-230 g) received a continuous infusion of isonitrogenous, isocaloric solutions with (+) or without (-) GLN. 5FU was administered in increasing doses (0, 100, 150 mg/kg i/p) to 24 animals at the start of PN (short-term) and to a further 16 animals (150 mg/kg) that had received PN for 5 days prior to treatment ( long-term). At the end of the experiment 4 days after 5FU, WBC, Hb, platelets and plasma GLN were measured, jejunal samples were weighed, examined for DNA and protein and histological sections were prepared. A portion of the results are shown (mean ± SEM,<*>p < 0.05,<**>p < 0.025 compared to -GLN).

5FU treatment decreased intestinal weight, DNA and protein. Rats that received GLN had significantly greater mucosal mass compared to animals that received standard solution. Addition of GLN was associated with increased survival in the long-term animals. Supply of GLN in nutrient solutions improves small intestinal cellularity and modifies the gastrointestinal toxicity of 5FU. Such therapy can reduce morbidity and mortality in patients whose gastrointestinal barrier defenses are damaged.

The specification has now been fully described and it will be clear to those skilled in the art that many changes and modifications can be made without affecting its spirit and scope.

Claims (18)

1. Method for treating catabolic dysfunction in an animal, characterized in that the animal is administered a therapeutically effective amount of glutamine or an analogue thereof in an amount greater than that present in the animal's normal diet. 2. Method according to claim 1, characterized in that the catabolic dysfunction is enteral or parenteral. 3. Method according to claim 2, characterized in that the enteral dysfunction essentially occurs in connection with intravenous feeding. 4. Method according to claim 3, characterized in that the enteral dysfunction affects the small intestine. 5. Method according to claim 2, characterized in that the parenteral dysfunction essentially occurs in connection with a physical trauma. 6. Method according to claim 5, characterized in that the physical trauma is associated with surgery, sepsis, burns, anorexia, chemotherapy, radiation therapy or uncontrolled diabetes. 7. Method According to claim 1, characterized in that the administration of a therapeutically effective amount of glutamine is enteral or parenteral. 8. Method according to claim 7, characterized in that the administration takes place using a dietary supplement. 9. Method according to claim 7, characterized in that the parenteral administration is by subcutaneous, intramuscular or intravenous injection, nasopharyngeal or mucosal absorption or transdermal absorption. 10. Method according to claim 8, characterized in that the enteral administration takes place in an amount of 0.3 to 2.0 g of glutamine or glutamine analogue per kg body weight per day. 11. Method according to claim 10, characterized in that the enteral administration takes place in an amount of 0.3 to 1.5 g of glutamine or glutamine analogue per kg body weight per day. 12. Method according to claim 11, characterized in that the enteral administration takes place in an amount of 0.4 to 1.0 g of glutamine or glutamine analogue per kg body weight per day. 13. Method according to claim 9, characterized in that the parenteral administration takes place in an amount of from 0.2 to 3.0 g of glutamine or glutamine analogue per kg body weight per day. 14. Method According to claim 13, characterized in that the parenteral administration takes place in an amount from 0.3 to 2.5 g of glutamine or glutamine analogue per kg body weight per day. 15. Method according to claim 14, characterized in that the parenteral administration takes place in an amount from 0.4 to 2.0 g of glutamine or glutamine analogue per kg body weight per day. 16. Mixture, characterized in that it comprises a glutamine-rich mixture to prevent or improve a catabolic dysfunction. 17. Container-for a liquid mixture which is adapted for Intravenous administration of the mixture, characterized in that it comprises a container for the mixture, and a fluid-conducting device connected to the container which can be attached to a needle for intravenous dispensing, where the mixture comprises an amount of glutamine which can inhibiting a catabolic dysfunction in an animal. 18. Container according to claim 17, characterized in that the catabolic dysfunction is associated with skeletal muscle loss.