Clinicai Nutrition (IYYZ) 11: X-215 0 Longman Group UK Ltd 1992

Effect of glutamine-supplemented total parenteral nutrition on the small bowel of septic rats

M. S. M. ARDAWI

Department of Clinical Biochemistry, College of Medicine and Allied Sciences, King Abdulaziz University, P.O. Box 9029, Jeddah 21413, Saudi Arabia (Correspondence to M.S.M.A.)

ABSTRACT-In order to study the effect of total parenteral nutrition (TPN) with or without glutamine supplementation in septic rats, septic Wistar albino rats were randomly assigned to receive 0.239 of nitrogen and 113kJ (1009 BW)-’ per day in the form of amino acids with (group 2) or without (group 1) glutamine supplementation or 10% (w/v) glucose only (group 3). After 4 days of TPN treatments, rats receiving glutamine-supplemented TPN had a cumulative nitrogen balance of -24.4 + 3.3mg N, which was significantly (P < 0.001) better compared to other TPN-treated groups. Septic rats of group 2 survived sepsis significantly (P < 0.001) better than those in groups 1 and 3. Glutamine-supplemented TPN treatment resulted in significant increases in jejunal weight (P < O.OOl), DNA and protein contents (P < O.OOl), villous height (P < 0.001) and crypt depth (P < 0.001) when compared with septic rats of group 1. Septic rats of group 2 extracted and metabolised glutamine bythe small bowel at higher rates (P < 0.001) than that observed in septic rats of group 1. Increases in jejunal glutaminase (38.2%, P < 0.001) and decreases in glutamine synthetase (41.7%, P < 0.001) activities were observed in response to glutamine-supplemented TPN treatment. It is concluded that the administration of glutamine- supplemented TPN is beneficial to the small bowel of septic rats.

Introduction

Sepsis which can be a complication of major trauma such as burns or abdominal surgery, is associated with profound metabolic abnormalities in carbohydrate, protein and lipid metabolism (for reviews see (1,2, 3)). The resulting septic compli- cations lead to conditions clinically described as a failure of major organ systems.

The small bowel mucosa is a dynamic tissue that is characterised by a high rate of cellular replica- tion and differentiation. Total parenteral nutrition (TPN) is associated with intestinal mucosal atro- phy (4, 5). Such changes are thought to be secondary to several factors including: the absence of luminal substrates (6), dietary amines (7), or fermentable fibres (8), changes in neurohormonal events (9), an&or absence of specific nutrients in currently used TPN formula (10). Therefore, maintenance of intestinal cellular growth and secretory and absorptive functions requires the supply of readily available fuel.

Glutamine is the most abundant amino acid in the body and plays a central role in inter-organ

nitrogen transport in normal and pathological states (for reviews see (11, 12)). The major site of utilisation of glutamine in the non-hepatic splanch- nit bed is the mucosa of the smaI1 bowel (for review see (13)). Most of the energy required by these cells is provided by the oxidation of glucose and glutamine in the ‘fed’ state and of glutamine and ketone-bodies in the ‘starved’ state (for review see (13)). Intestinal glutamine utilisation and metabolism is increased following surgery (14), thermal injury (15) or glucocorticoid treatment (16), but is decreased in response to sepsis (17).

Commercially available amino acid solutions that are used in the formulation of parenteral nutrient mixtures do not contain glutamine. Recent studies showed that supplementation of TPN solutions with glutamine attenuates mucosal atrophy related to TPN treatment, and improves small-bowel cellularity of injured mucosa after chemotherapy and radiation treatment (for review see (18)). However, the effects of administration of TPN-supplemented glutamine on the small bowel of septic rats (sepsis was induced by caecal ligation and puncture) is unknown. The purpose of

208 INTESTINAL GLUTAMINE AND TPN IN SEPTIC RATS

this study was to investigate the effects of adminis- tering to septic rats glutamine-supplemented TPN on the small bowel.

Materials and methods

Animals This study was conducted in accordance with the national Institutes of Health guidelines for the use of experimental animals. Male Wistar albino rats (230-250g) (The animal house of King Fahd Medical Research Center, College of Medicine and Allied Sciences, King Abdulaziz University, Jeddah, Saudi Arabia) were housed in a controlled environment (constant temperature 24°C and a light cycle of 12h on/l2h off). Rats were main- tained on a standard diet (commercial rat cubes containing (w/w) approximately 18% protein, 3% fat, 77% carbohydrate and 2% organic salt mix- ture with a vitamin supplement; Grain Silos and Flour Mills Organization, Jeddah, Saudi Arabia) and water ad libitum. Animals were started on TPN 2 h after the operation for induction of sepsis and catheterisation (see below).

Chemicals and enzymes Enzymes and coenzymes were obtained from Boehringer Mannheim GmbH (Mannheim. Ger- many). Chemicals for liquid scintillation measure- ment of radioactivity were obtained from Fisons Scientific Apparatus (Loughborough, Leics, UK). Radioactive materials were obtained from Amersham International (Amersham, Bucks, UK). Amino acids for intravenous solutions were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The vitamin mixture was obtained from Armour Pharmaceutical Co. (Blue Bell, PA, USA). All other reagents of analytical grade were obtained from BDH Chemicals (Poole, Dorset, UK) or May and Baker (Dagenham, Essex, UK).

Preparation of TPN solutions Intravenous solutions were designed and prepared to provide the nutritional requirements for growth in rats (see (19)). Prepared solutions were mixed under a laminar flow hood (pbi International, Milano, Italy) to maintain optimal sterile con- ditions and were further sterilised using Millipore membrane filters (size 0.22pm; Millipore Corp., Bedford, MA, USA). Prepared solutions were stored at 4°C until the end of the 4-day infusion period. Checks were made on nitrogen and gluta- mine concentrations in prepared solutions

(nitrogen concentrations were found to be within 1.5-2.3% of anticipated values). Every day, new 50ml syringes (Beckton-Dickinson and Co., Rutherford, NJ, USA) were filled from the refrigerated stock solution and the parenteral nutrients were administered with an Ivac syringe- pump (model 700; Ivac Corp., San Diego, CA, USA). Stability studies demonstrated that the degradation of glutamine in prepared solutions was undetectable when stored at 4°C for 5 days and was 0.15% per 24h when stored at room temperature.

Treatment of animals A sterile silastic catheter (internal diameter 0.75mm, medical grade) was inserted into rats under sodium pentobarbital anaesthesia [40mg (kg BW)-‘1 through the external jugular vein, and secured in place at the entry site. Tunnelled subcutaneously, the tubing exited through a stab wound in the skin of the mid-scapular region and was secured to the deep fascia by using a stainless- steel button; it was protected by a coiled cable connected to a swivel and harness, allowing free movement. After anaesthesia and catheterisation, sepsis was induced by means of a caecal ligation and puncture technique (see (17)) and rats received saline [0.9% (w/v) NaCl; 0.25ml (kg SW))‘] subcutaneously and were returned to individual metabolic cages that allowed the separate collection of urine and faeces. For the following 2h, each animal received an intravenous infusion of sahne [31ml (1OOg BW)-’ per day]. The animals were then randomly assigned to groups to receive one of three different TPN solutions for a 4-day period. Group 1 (n = 14) received a conventional hyperalimentation amino acid solution, (conventional TPN) and group 2 (n = 14) received a glutamine-supplemented TPN hyperalimentation solution. The two TPN solu-

tions were isocaloric and isonitrogenous with each other (see Table 1). The major nutritional difference between them was the quantity of glutamine. This was added at 2% (w/v) (18); however, in order to achieve isonitrogenous and isocaloric formulae in the presence of glutamine, the non-essential amino acid component was modified (see Table 1). The daily dosages were approximately 0.23 g of nitrogen per 1OOg BW and 113kJ per 1OOg BW. A third group of septic rats (group 3, n = 20) received a solution without amino acids but containing 10% (w/v) glucose in saline at 113kJ per 1OOg BW. Group 3 was included as a reference group together with a

CLINICAL NUTRITION 209

1OOg SW) via a tertiary branch of the mesenteric vein. After 30 min of equilibration. three consecu- tive 10 min collection periods were observed, and arterial (abdominal aorta) and venous (portal vein) blood was collected for the determination of the concentration of PAH. Intestinal blood flow was calculated according to the equation:

PAH administered

Blood flow (mg min-‘)

(ml min-‘) = venous [PAH] (mg ml-‘) arterial [PAH] (mg ml-“)

The concentration of PAH was measured in a Somogyi filtrate [lo% (v/v) barium hydroxide and zinc sulphate], which was treated with p-dimethyl- aminobenzaldehyde alcohol solution as described previously (22). Net rates of exchange of gluta- mine, glutamate, alanine and ammonia were calculated by multiplying intestinal blood flow by the respective arteriovenous concentration difference.

Preparation of homogenates and assay of enzyme activities

Animals were killed by cervical dislocation. The small intestine (from the duodenum to the cae- cum) was rapidly removed, stripped of its mesen- tery, washed by forcing ice-cold 0.9% (w/v) NaCl through the lumen, and 1Ocm lengths of jejunum were measured against a vertical scale with a 5g stretch. Jejunal segments were placed on a glass plate over ice, split longitudinally. The first 3cm were used for histological studies (see below) and the next 7cm were used for enzyme activity measurements and analysis of DNA and protein contents (see below). Intestinal mucosa was separated from the underlying muscle by scraping with a microscope slide, weighed and homoge- nised in 5-10 vol of extraction media (for glutami- nase (23) and glutamine synthetase (24), respectively) by using a Polytron homogeniser PCU-2 (Kinnematica GmbH, Kriens-Lucerne, Switzerland) at position 4 for 10-20s at 0-4°C. The whole homogenate was used for enzyme assays. Both the activities of glutaminase and glutamine synthetase were assayed radiochemically as described previously (see (24, 25)).

Table 1 Composition of amino acid solutions used

Amino acid

Amino acid concentration (g/l)

Without glutamine With glutamine

Alanine Aspartate Arginine Cysteine Glutamate Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

4.5 3.6 4.4 0.1 0.3 0.0

13.3 2.1 1.7 3.3 2.9 1.2 2.8 3.1 1.4 1.4 0.5 0.1 2.0

2.4 1.2 2.6 0. I 0.3

20.0 0.9 2.1 1.7 3.3 2.9 1.2 2.8 1.0 0.4 1.4 0.5 0. 1 2.0

Vitamins and salts were added at the usual dosages: ascorbic acid (lOOmg), vitamin A (3300 i.u.). vitamin D (200 i.u.). thiamine (3.0mg), riboflavin (3.6mg), pyridoxine HCI (4.0mg). niacinamide (40.0mg), pantothenic acid (15.0mg). vitamin E (10 i.u.), biotin (6Opg). folic acid (4OOpg). vitamin Biz (5 pg). potassium chloride (447mg), sodium chloride (61.3mg). sodium acetate (721.6mg), calcium gluconate (17.8mg). potassium phosphate (2.8mg) and magnesium sulphate (2.0mg).

control chow-fed nonseptic group (group 4, n = 10) for comparative purposes. TPN solutions were administered for 4 days at a flow rate of 48ml per day as described above, and animals were not permitted access to their normal diet during the TPN period.

Measurement of arteriovenous-concentration difference and blood flow Rats of groups 1,2, and 4 were anaesthetised with ether, and blood was withdrawn simultaneously into heparinised syringes from the hepatic portal vein and the abdominal aorta. Samples (l.O- 1.5 ml) were quickly added to 0.1 ml of ice-cold HCI04 (lo%, v/v) and were used for the determin- ation of concentrations of metabolites after deproteinisation and neutralisation (see (20)).

Intestinal blood flow was measured by the indicator-dilution technique described by Katz and Bergman (21). p-Aminohippurate (PAH), a non-metabolisable dye, was used to measure intestinal blood flow by the dye-dilution tech- nique. PAH was administered (3OFg min-’ per

Determination of concentrations of metabolites and nitrogen balance Concentrations of metabolites in neutralised extracts of plasma were determined spectropho-

210 INTESTINAL GLUTAMINE AND TPN IN SEPTIC RATS

tometrically with a Beckman DU-6 recording spectrophotometer (Beckman Instruments, Full- erton, CA, USA) by standard enzymic methods, as described previously (20).

Nitrogen balance measurements were made over the 4-day TPN treatment as previously described (see (17)).

Protein and DNA contents in jejunal mucosa were measured by the methods of Lowry et al (26) and Burton (27), respectively.

Histological examination Animals were killed by cervical dislocation, and jejunal samples were isolated as described above. Isolated tissues were flushed with cold saline (0.9% w/v, NaCl) and used for histological exam- ination. For histological evaluation, tissues were fixed in 10% (v/v) formalin, embedded in paraffin, and stained with haematoxylin-eosin. Villus height and crypt depth were determined on tissue cross- sections by using an ocular micrometer. Measure- ments were carried out in a blind fashion on coded slides. All measurements were made in triplicate and a mean value was obtained.

Statistical analysis Data are presented as mean k SEM for each group of animals studied. The survival rates of the septic groups were compared by x2 analysis. Statistical analysis were performed by analysis of variance with Scheffe F-test. Differences were considered statistically significant at a P value of <0.05.

Results

Survival rate and nitrogen balance in septic rats

The group receiving glutamine-supplemented TPN (i.e. group 2) survived sepsis significantly better than did the rats in group 1 (P C 0.001) over the 4 days of TPN treatment after caecal ligation and puncture, The cumulative percentage of deaths in group 2 was 21.4% (3 deaths out of 14 rats) over 4 days of observation compared with 42.9% (6 deaths out of 14 rats) and 70% (14 deaths out of 20 rats) in rats of groups 1 and 3, respectively.

The Figure shows the daily cumulative nitrogen balance data (mg N) over the 4-day observation period. Progressive deterioration by sepsis added to surgical stress produced a negative nitrogen balance which is consistent with previous studies (e.g. (17, 20)) and related to the severity of the

+ 20

-120

DAYS OF TREATMENT

3

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Fig. Cumulative nitrogen balance during the 4 days of treat- ment. 0, Group 1; k?3, group 2; 8. group 3. Values are means with bars indicating SEM.

catabolic state of animals. On day 1 after treat- ment, apparent nitrogen balance equalled -16.2 2 2.2mg N in rats receiving TPN without gluta- mine (group l), - 15.6 + 2.8mg N in rats receiving TPN with glutamine (group 2) and -34.5 + 3.7mg N in rats receiving glucose (group 3). Group 1 and 2 did not differ significantly. Cumulative nitrogen balance in group 1 reached -50.6 + 5.0mg N on day 2, more negative (P < 0.001) than the gluta- mine-supplemented TPN group 2, with -25.0 + 2.4mg N. Cumulative nitrogen balance in groups 1 and 2 was better compared with the glucose group (P < 0.001). Cumulative nitrogen balance on day 3 equalled -73.6 + 7.3mg N in rats of group 1, -29.6 & 2.7mg N in rats of group 2 and -86.2 * 3.0mg N in rats of group 3. On day 4 only rats of group 2 receiving glutamine-supplemented TPN had a positive nitrogen balance with the least negative cumulative nitrogen balance of -24.4 + 3.3 mg N compared to that of rats of group 1 (being -84.0 k 9.lmg N) and group 3 (being -105.9 f 9.5 mg N) respectively.

Intestinal morphology Animals receiving intravenous feeding exhibited decreases in intestinal weight, DNA, protein con- tent, villus height and crypt depth (Table 2). Jejunal wet weight in rats receiving glutamine-sup- plemented TPN was 42.1 ? 4Smg cm-’ vs 32.5 f 3.5mg cm-’ in rats receiving TPN without gluta- mine (P < 0.001). There was a concomitant increase in DNA (by 28.1%) and protein (by 30.8%) content in rats receiving glutamine-sup- plemented TPN when compared to those of group 1 receiving TPN without glutamine (Table 2). Morphometric analysis of the intestine confirmed such changes: the villi in the jejunum were signifi- cantly longer in rats receiving glutamine-sup- plemented TPN (by 21.7%) as compared to that of rats receiving TPN without glutamine (P < 0.001). Jejunal crypt depth was increased by about 23.0% (P < 0.001) in response to glutamine-supplemen- ted TPN (Table 2).

Arteriovenous-concentration differences and net rates of utilisation of glutamine The arterial concentration of glutamine was increased by approximately 41.9% in rats receiv- ing TPN with glutamine compared to rats receiving TPN without glutamine (Table 3). Blood ammonia levels increased slightly but not significantly from 32 + 5 to 37 + 7 nmol ml-’ in rats receiving TPN without glutamine and TPN with glutamine, respectively.

Arteriovenous-concentration differences across the small bowel showed a net uptake of glutamine, with a net release of glutamate, alanine and ammonia (the major nitrogenous end-products of glutamine metabolism) (Table 3). Rats receiving glutamine-supplemented TPN exhibited marked increases in the arteriovenous-concentration difference for glutamine (72.4%), glutamate

CLINICAL NUTRITION 211

(113.2%), alanine (75.0%) and ammonia (21.4%) (Table 3) when compared to rats receiving TPN without glutamine, respectively (see Table 3).

Calculation of the rates of utilisation or metabo- lite production from substrate arteriovenous- concentration differences and bloodflow data indi- cated an increased rate of utilisation of glutamine (by 75.9%) and production of glutamate (by 113.1%), alanine (by 75.4%) and ammonia (by 26.0%) in rats receiving glutamine-supplemented TPN when compared to those receiving TPN without glutamine, respectively (Table 3).

Jejunal activities of glutaminase and glutamine synthetase Jejunal glutaminase activity was increased (by 38.2%) in response to the infusion of TPN with glutamine (Table 4). Jejunal glutamine synthetase activity was significantly higher (P < 0.001) in rats receiving TPN without glutamine (Table 4).

Discussion

Following sepsis (and other catabolic states), cur- rent therapies are directed towards improving nitrogen sparing in septic patients. However, administration of high dosages of nitrogen failed to show beneficial effects on nitrogen balance in septic patients (28,29). The use of branched chain amino acids supplemented with lipids (as an energy source) are reported to have some effect on nitrogen sparing (both in septic patients and septic rats), but this is still controversial (30,31,32). The important finding in the present study is that the administration of a glutamine-supplemented TPN solution to septic rats resulted in: 1) improved nitrogen balance and survival rates, 2) increased intestinal mucosal thickness, DNA and protein

Table 2 Jejunal morphology. DNA content and protein content of septic rats of groups 1 ,2 and chow-fed control (group 4) at 4 days after operation

TPN without glutamine TPN with glutamine Chow-fed control (group 1, n = 9) (group 2, n = 11) (group 4, n = 10)

Jejunal wet weight (mg cm-‘) 32.5 f 3.5 42.1 f 4.5* 52.8 + 2.7 Jejunal DNA (p,g cm-‘) 228 + 27 292 + 20* 345 Ifr 23 Jejunal protein (mg cm-‘) 3.18 + 0.27 4.16 f 0.30* 5.55 + 0.42 Protein/DNA ratio (mg mg-‘) 13.9 14.2 16.1 Villus height (wm) 507 f 15 617 z!x 25* 739 + 24 Crypt depth (urn) 113 f 10 139 f 9 158 + 8 Villuskrypt ratio 4.5 4.4 4.7

Data are mean + SEM for (n) being the number of animals used. Group 4 was included for comparison only. * P 0.001 1 < vs group

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CLINICAL NUTRITION 213

Table 4 Maximal activities of glutamine synthetase and glutaminase in jejunal mucosal scrapings of septic rats of groups 1 and 2 and chow-fed (group 4) rats at 4 days after operation

Treatment

group

TPN without glutamine (Group 1. n = 9)

TPN with glutamine (Group 2, n = 11)

Chow-fed (Group 4, n = 10)

Glutamine synthetase Glutaminase [f mol min-’ (kg DNA)-‘] [nmol min-’ (pg DNA)-‘]

338 f 49 34 * 6

197 + 19* 47 k 5”

208 527 67 * 9

Data are mean f SEM for (n) being the number of rats used. Group 4 was included for comparison only. * P < 0.001 vs group 1

content, and 3) increased net extraction and metabolism of glutamine by the small bowel, as compared with septic rats treated with TPN with- out the addition of glutamine. Other studies have demonstrated a beneficial effect of glutamine-sup- plemented TPN on nitrogen balance in experimen- tal animals after laparotomy (33), sepsis (34), and in patients after cholecystectomy (35). Moreover, the present study confirms the previous observa- tion in rats that atrophy of the intestinal mucosa is associated with intravenous feeding (see (4, 5, 36)). The decrease in intestinal cellularity has been partly attributed to a decrease in gastrointestinal secretions and peptide hormones release in the absence of luminal feeding (see (37, 38)). Gluta- mine administration to experimental animals was also reported to enhance mucosal hyperplasia after small bowel resection (39), preserve intesti- nal mucosa during parenteral nutrition (18). and improve small bowel cellularity after chemo- therapy (40) or radiation treatment (41).

The mechanism(s) by which glutamine exerts its effect on the intestinal mucosa of septic rats (as described herein) are unclear, however several possibilities are suggested. First, glutamine may have had direct cytoprotective effects on the cells of the small bowel: the small bowel of septic rats (without TPN treatment) exhibits decreased rates of glutamine consumption and metabolism (see (17)). Therefore, provision of glutamine could help to improve the maintenance of the gut cellularity and function via the supply of nucleo- tide bases and energy. Indeed, the provision of glutamine to septic rates was associated with increased villous height, crypt depth, DNA and protein contents (see Table 2), together with elevated rates of extraction and metabolism of the amino acid (see Table 3). These effects are further supported by the findings of Beaulie et al that

glutamine promotes cellular differentiation in intestinal cells in vitro (42). Secondly, glutamine may have had cytoprotective effects on the immune and repair cells: many specialised cells participate in intestinal immunity including intra- epithelial lymphocytes, macrophages and natural killer cells (43). Lymphocytes, macrophages and fibroblasts require glutamine for optimal immune response and cellular repair (see (44)). Therefore, the provision of glutamine could enhance the host’s immune defence mechanism(s) and improve cellular repair processes by supplying sufficient glutamine to satisfy the metabolic need for the amino acid by the immune cells and fibroblasts. Indeed, glutamine-related increases in levels of secretory immunoglobulin A with subsequent reduced adherence of enteric bacteria to the intestinal mucosa (43) further support the role of glutamine in small bowel immunity (see also (44)). Thirdly, glutamine could serve as a secretagogue stimulating the elaboration of peptide hormones (e.g. enteroglucagon) which exert trophic effects on the mucosa (45).

In septic rats treated with glutamine-sup- plemented TPN (as described herein), the plasma glutamine concentrations never exceeded 30--40% of basal levels (Table 3). The latter suggests that adaptive responses must have occurred to facil- itate the clearance of glutamine from the circu- lation ’ septic rats treated with glutamine?upplemented TPN. Indeed, the small bowel of glutamine-supplemented TPN septic rats extracted and metabolised glutamine at higher rates with concomitant production of alanine, glutamate and ammonia than that obtained in corresponding controls (Table 3). Alanine could be used for hepatic gluconeogenesis (see (46)). The increase in small bowel glutamine extraction was accompanied by an increase in the specific

214 INTESTINAL GLUTAMINE AND TPN IN SEPTIC RATS

activity of glutaminase (Table 4): this may be an adaptive response designed to ensure adequate metabolism of circulating (and/or luminal) gluta- mine in the small bowel. Possibly this response is also necessary to provide carbons for the tricar- boxylic-acid cycle intermediates and nitrogens for DNA biosynthesis. Moreover, the activity of intestinal glutamine synthetase was decreased in response to glutamine-supplemented TPN: this may be due to a decrease in the rate of the local synthesis of glutamine when the amino acid is provided via TPN treatment.

In summary, the administration of glutamine- supplemented TPN proved to be beneficial to septic rats and resulted in increased jejunal muco- sal weight, DNA and protein content, and villus height together with greater extraction and meta- bolism of glutamine by the small bowel. Improved mucosal cellularity of the small bowel may improve digestive function, maintain intestinal barrier function and facilitate introduction of enteral nutrition. Therefore, patients with sepsis may benefit from the addition of glutamine to their nutritional therapy.

Acknowledgements

This work was supported by university grants to M.S.M.A. 026/409 at the Clinical Metabolic Research Laboratory at King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia.

I thank Miss Sanna Kateilah. Mrs Shiba Ansari and Mr Peter Lacy for their technical help, and Mrs Helen Khoja for her excellent secretarial assistance.

References

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Submission date: 12 September 1991; Accepted after revision: 23 April 1992