Neither glutamine nor arginine supplementation of diets increase glutamine body stores in healthy growing rats

Clinical Nutrition (2000) 19(5): 319–325 # 2000 Harcourt Publishers Ltd doi:10.1054/clnu.2000.0115, available online at http://www.idealibrary.com on

ORIGINAL ARTICLE

Neither glutamine nor arginine supplementation of diets increase glutamine body stores in healthy growing rats J. J. BOZA, D. MOEºNNOZ, A. R. JARRET, J. VUICHOUD, C. GARCIØ A-ROØDENAS, P. A. FINOT, O. BALLEØVRE Nestle¤ Research Center, Nestec Ltd., Lausanne, Switzerland (Correspondence to: JJB, Nestle¤ Research Center, Nestec Ltd, Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland)

AbstractöThe aim of the work was to resolve whether glutamine and arginine supplemented diets a¡ect plasma and tissue (muscle, liver and intestinal mucosa) glutamine concentrations, as well as glutaminase and glutamine synthetase speci¢c activities.The trial was performed in growing rats fed 10% protein diets for 3 weeks. Protein sources were: whey proteins (W); whey proteins þ free glutamine (WG); whey proteins þ arginine (WA); and casein þ wheat protein hydrolysate þ acid whey (39:39:22), as source containing protein-bound glutamine (CGW). Rats fed the control diet (6.4% glutamine) (W) showed comparable glutamine body stores to those of rats fed the WG diet. In fact, glutamine supplementation down-regulated the hepatic glutamine synthetic capacity of growing rats (W/WG: 6.8 + 0.3 vs 6.0 + 0.2 nmol/min/mg protein). Arginine supplementation of the diet (up to 9% of the protein content) resulted in a decrease in plasma and tissue glutamine concentrations (W/WA: plasma, 1218 + 51 vs 1031 +48 mmol/L; liver 7.5 + 0.4 vs 6.5 + 0.2 mmol/g; muscle: 5.7 + 0.2 vs 4.0 + 0.2 mmol/g).These data suggest that glutamine supplementation of the diet does not increase plasma and tissue glutamine concentrations in healthy growing rats, while the addition of arginine to the diet decreases glutamine body stores. & 2000 Harcourt Publishers Ltd.

in these situations, the intracellular pools of glutamine in muscle are markedly reduced (4). Next, as tissue stores become depleted, plasma or whole blood glutamine concentrations fall. In recent studies, it has been shown that the addition of glutamine to parenteral and enteral formulas increased concentrations of this amino acid in blood, improving nitrogen balance, cell proliferation, incidence of infection, length of hospital stay, etc. (5–8). Tracer studies using stable isotopes have been done to gain more insight into the response of glutamine metabolism to exogenous glutamine. These studies have been conducted in human volunteers to calculate the incidence of oral glutamine on the rate of appearance of glutamine in plasma, coming from the de novo synthesis or released via the breakdown of proteins (9, 10). However, to our knowledge, there is no much evidence about how oral glutamine supplementation affects glutaminase and glutamine synthetase activities, as well as glutamine stores, other than those in skeletal muscle. The major objective of the present work was to resolve whether the content of glutamine (free or bound to protein) and arginine in the diet affects plasma and tissue (skeletal muscle, liver and intestinal mucosa) glutamine concentrations, as well as the specific activities of glutaminase and glutamine synthetase in liver, skeletal muscle and gut mucosa of growing healthy rats.

Key words: glutamine; arginine; enteral nutrition; de novo synthesis of glutamine; whey proteins; rats

Introduction Historically, glutamine has not been used as a nutritional supplement because it is considered a nonessential amino acid. However, glutamine plays a major role in important biochemical pathways. It is the preferred energy source for rapidly dividing tissues, essential precursor for nucleotide synthesis, a constituent amino acid in synthesis of body proteins, it contributes to the interorgan nitrogen and carbon transport, etc. (1). Glutamine is the most abundant amino acid in the body, contributing to 450% of the free amino acid pool in human skeletal muscle, while glycine is the most abundant in rat skeletal muscle (2). Under physiologic conditions, sufficient amounts of glutamine are produced endogenously to allow the maintenance of body’s glutamine stores (especially the skeletal muscle) and to cover the demands of the glutamine-consuming tissues (3). In humans, during the stress associated with injury, sepsis and inflammation, there is a marked increase in glutamine consumption by the gastrointestinal tract, immunologic cells, inflammatory tissue, and kidney. Requirements for glutamine by these tissues may outstrip the synthetic capacity of the skeletal muscle. Thus, 319

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GLUTAMINE AND ARGININE ON GLUTAMINE BODY STORES

Materials and methods Groups and diets Acid whey was used as control protein source rich in branched chain amino acids, supplemented or not with glutamine (total content in protein mixture: 15 g/100 g protein) or arginine (total content in protein mixture: 9 g/100 g protein). To study the influence of the molecular form of the glutamine, wheat protein hydrolysate was used as source of protein bound glutamine. Wheat protein hydrolysate was mixed with casein and acid whey to balance essential amino acid composition. Diets were isonitrogenous and contained 10% protein. The chemical composition of the four different diets is given in Table 1. The protein sources of the different diets were as follows: Acid whey: Acid whey þ glutamine (15% of total amino acids): Acid whey þ arginine (9% of total amino acids): Casein þ wheat protein hydrolysate þ acid whey (39:39:22):

W WG WA

Table 2 Amino acid composition of protein mixtures (g/100 g protein)

CGW

Amino acid

W*

WG*

WA*

CGW*

Asp Glu Ser Gly Gln** His Thr Ala Arg Pro Tyr Val Met Cys lle Leu Phe Trp Lys S Essential S BCAA

9.96 10.16 5.09 2.56 6.67 2.12 4.45 5.52 3.18 5.68 2.32 4.19 2.62 3.14 5.33 12.36 3.63 2.27 10.52 50.83 21.88

8.88 8.92 4.79 2.43 14.45 1.94 4.30 4.81 2.53 5.46 1.57 3.52 2.44 2.87 5.31 12.09 3.49 2.01 9.83 47.43 20.92

9.61 8.60 4.85 2.51 6.97 2.03 4.42 5.03 8.97 5.63 1.88 3.52 2.43 2.88 4.99 12.07 3.46 2.06 9.62 47.33 20.58

6.94 7.13 5.21 2.82 14.41 2.26 3.64 4.01 3.15 10.69 2.21 3.91 2.51 1.71 4.69 10.78 4.96 1.60 7.64 43.65 19.38

Diets WG and CGW had the same glutamine content. Acid whey protein concentrate and casein were supplied by MD Food Ingredients (Videbaek, Denmark), glutamine and arginine were supplied by Ajinomoto (Tokyo, Japan) and the wheat protein hydrolysate was provided by DMV International (Veghel, Netherland).

Experimental design Thirty-two male Wistar rats, at weaning, were obtained from Iffa Credo (France). Animals were allocated to individual cages and were maintained at 238C with a 12h light-dark cycle. They received the whey protein based diet (Table 3 shows its chemical composition) ad libitum for 3 days. Then, animals were randomized to study

Table 1 Chemical composition of diets (g/100 g) Ingredient Protein mixture W* Protein mixture WG* Protein mixture WA* Protein mixture CGW* Corn oil Corn starch Sucrose Cellulose Inositol Choline bitartrate AIN–76A Mineral mix AIN–76A Vitamin mix

groups by weight. Eight animals were kept on the same diet (whey protein based diet) for the rest of the study (group W), whereas the remaining animals were divided in three groups of eight animals each and fed ad libitum one of the other three diets (WG, WA, and CGW). From this moment, diet consumption was controlled for 3 weeks. During the second week, animals were transferred to metabolic cages and kept on the same diet ad libitum. During this period, faeces and urine from all rats were collected to determine N balance. After the three-week period, diets were removed from the cages and animals were sacrificed under anaesthesia, after an overnight fast. The sacrifice proceeded as follows: rats were under isoflurane anaesthesia and total bleeding was performed in dorsal aorta on heparinized tubes, immediately centrifuged for 10 min at 2000 g at 48C to obtain plasma. The liver, muscle tibialis and the small

W

WG

WA

CGW

13.88 – – – 7.05 59.35 10.00 5.00 0.03 0.20 3.50 1.00

– 13.77 – – 7.10 59.41 10.00 5.00 0.03 0.20 3.50 1.00

– – 13.79 – 7.10 59.39 10.00 5.00 0.03 0.20 3.50 1.00

– – – 12.42 7.60 60.26 10.00 5.00 0.03 0.20 3.50 1.00

*W: Acid whey. *WG: Acid whey þ glutamine (15% of total amino acids). *WA: Acid whey þ arginine (9% of total amino acids). *CGW: Casein þ wheat protein hydrolysate þ acid whey (39:39:22).

*W: Acid whey. *WG: Acid whey þ glutamine (15% of total amino acids). *WA: Acid whey þ arginine (9% of total amino acids). *CGW: Casein þ wheat protein hydrolysate þ acid whey (39:39:22). **Glutamine content was measured after extensive enzimatic hydrolysis. BCAA: branched chain amino acids.

Table 3 Tissue glutamine concentration of rats fed the different experimental diets (mmol/g tissue) (mean + SEM, n=8) Diet W* WG* WA* CGW*

Jejunum mucosa

Liver

Muscle tibialis

3.3 + 0.3 2.6 + 0.4 3.0 + 0.6 2.8 + 0.7

7.5 + 0.4a 7.9 + 0.3a 6.5 + 0.2b 7.5 + 0.3a

5.7 + 0.2a 5.5 + 0.2a 4.0 + 0.2b 4.4 + 0.3b

Values not sharing the same letter are significantly different (P50.05). *W: Acid whey. *WG: Acid whey þ glutamine (15% of total amino acids). *WA: Acid whey þ arginine (9% of total amino acids). *CGW: Casein þ wheat protein hydrolysate þ acid whey (39:39:22).

CLINICAL NUTRITION

intestine were also collected. Jejunum samples were scraped with a glass slide under ice, to obtain the jejunum mucosa. The liver, the muscle sample and the jejunum mucosa were stored at 7808C until analysis. Growth test The indices used to estimate the nutritional quality of the protein sources were determined as follows (11): . . . . .

Body weight gain (g/3 weeks) Digestibility: absorbed N/ingested N Biological value (BV): retained N/absorbed N Net protein utilization (NPU): retained N/ingested N Protein efficiency ratio (PER): body weight gain (g/3 weeks)/ intake of proteins (g/3 weeks).

Plasma amino acids Once obtained, 200 mL of plasma were deproteinized by addition of 20 mL of a solution containing sulfosalicylic acid (400 g/L) and vitamin C (60 g/L). After mixing, samples were centrifuged (10000 g 3 min) and supernatants were frozen at 7808C until analysis, after the addition of internal standards (100 mM D-glucosaminic acid and 100 mM S-(2-aminomethyl)-L-cysteine.HCl). The analyses were performed in a Beckman 6300 amino acid analyser (Palo Alto, USA). To avoid glutamine degradation, samples were kept at 108C before injection (12). Glutamine content of muscle, liver and jejunum mucosa Two hundred mg of tissue (muscle, liver and jejunum mucosa) were mixed with 2 mL of perchloric acid solution (5% w/v) and homogenized with the aid of a Polytron at 10000 rpm for 1 min. Samples were then centrifuged at 13000 g (17000 g for liver samples) 10 min. at 48C. Supernatants were filtered using membranes with a molecular weight cut-off of 10000 Da and filtrates were stored at 7808C until analysis. The analysis was performed by reversed-phase HPLC, after derivatization with phenylisothiocyanate. These derivatives were separated on an octadecylsilyl reversed-phase column. The elution solvent consisted of solvent A, containing 70 mmol/L sodium acetate in water (pH:6.45), and solvent B, containing 450 g/L acetonitrile and 150 g/L methanol in water. The gradient mixing was as follows: 0–13.5 min, 3% B, 13.5–14 min, 100% B, 14–34 min 100% B, 34–35 min 0% B, 45 min 0% B. The column effluent was monitored spectrophotometrically at 254 nm (13). Liver and jejunum mucosa glutaminase activity Two hundred mg of jejunum mucosa or 400 mg of liver were mixed with 2 mL of a buffer containing 300 mM mannitol and 5 mM Hepes, pH 7.4 and homogenised

321

with the aid of a Polytron at 10000 rpm for 1 min. Then, samples were centrifuged at 500 g 10 min at 48C. Supernatants were further centrifuged at 5000 g to isolate mitochondria, according to Szweda & Atkinson (14). Mitochondria were then dissolved in 1 mL water. An aliquot (100 mL) was added to 100 mL of a mixture containing 100 mM potassium phosphate, 171 mM glutamine, 1.5 mM ammonium chloride and 10% ethyleneglycol, pH 8.0 and were incubated at 378C for 30 min. The reaction was terminated by adding 40 mL of TCA solution (7% w/v), kept on ice for 15 min and centrifuged at 10000 g 2 min (15). The supernatant was then assayed for glutamate determination, following a colorimetric method (Boehringer Mannheim, Germany) (16). Glutaminase activity was expressed in nmol of glutamate produced per minute per mg mitochondrial protein. Mitochondrial protein was measured using the bicinchoninic acid method kit (Pierce, US) (17). Liver and skeletal muscle glutamine synthetase activity Four hundred mg of liver of 100 mg muscle tibialis were mixed with 2 mL of a buffer containing 50 mM imidazole-HCl buffer (pH 6.8) and homogenised with the aid of a Polytron at 10000 rpm for 1 min. After, samples were centrifuged at 4500 g 15 min. at 48C. Glutamine synthetase activity was determined in the supernatant by measuring the formation of g-glutamylhydroxamate when hydroxylamine is substituted for ammonia, as described by Minet et al. (18). Briefly, 100 mL of supernatant was added to 0.9 mL of a solution at 378C containing imidazole-HCl buffer, pH 7.2 (50 mmoles), MnCl2 (20 mmoles), 2-mercaptoethanol (25 mmoles), sodium L-glutamate (50 mmoles), hydroxlamine (100 mmoles), and sodium ATP (10 mmoles). After incubation at 378C for 15 min, 1.5 mL of a solution containing 0.37M FeCl3, 0.67M HCl and 0.2M TCA, was added. The precipitated protein was removed by centrifugation, and the optical density at 535 nm of the solution read against a reagent blank containing the total incubation mixture except for the homogenate and ATP. Under these conditions, 1 mmol of g-glutamylhydroxamate gave an optical density of 0.34. The rate of the enzymatic reaction was linear with enzyme concentration over the range 0.2–1.2 mmoles of g-glutamylhydroxamate formed. The activity of glutamine synthetase was expressed in nmol g-glutamylhydroxamate formed per minute per mg of protein. Tissue protein was measured using the bicinchoninic acid method kit (Pierce, USA) (17). Statistical analysis Data are expressed as mean + SEM. One-way analysis of variance and post hoc Bonferroni tests were used to determine mean differences among the groups for all the parameters studied. A difference was considered significant at P5 0.05.

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GLUTAMINE AND ARGININE ON GLUTAMINE BODY STORES

Results There were no differences in body weight gain, liver weight, protein intake, protein efficiency ratio and digestibility during the feeding period between groups (Table 4). Rats fed diets containing wheat protein hydrolysate exhibited the lowest values of nitrogen protein utilisation and biological value, as compared to those fed the other experimental diet. The supplementation of the protein source (acid whey) with either glutamine or arginine did not lead to any significant change in any parameters concerning body growth and nitrogen balance. The plasma amino acid profile of rats fed the acid whey based diet (W), the same dietþglutamine (WG) and the same dietþarginine (WA) was very similar. In fact, there were no differences in any amino acid concentrations between rats fed the diet W and those fed WG. Interestingly, glutamine supplementation of the diet did not lead to an increase in the plasma glutamine levels after 3 weeks of diet consumption. However, rats fed the diet WA showed significantly (P50.05) higher plasma concentrations of urea and lysine and lower concentrations of glutamine compared to those of rats fed the control diet or the control diet plus glutamine (Table 5). Table 3 shows the glutamine concentration in jejunum mucosa, liver and muscle tibialis of rats fed the different experimental diets. There were no significant differences in the glutamine content of jejunum mucosa among groups. The addition of free glutamine to acid whey did not lead to a significant increase in muscle nor liver Table 4 Body weight gain, liver weight, food intake, protein intake, protein efficiency ratio, digestibility, net protein utilization and biological value during the feeding period of rats fed the experimental diets (mean + SEM, n=8) Parameter Weight gain (g/22 days) Liver weight (g) Protein intake (g/22 days) PER** Digestibility (%)** NPU (%)** BV (%)**

W*

WG*

WA*

CGW*

123 + 7

119 + 5

125 + 5

111 + 4

6.5 + 0.3 38.1 + 1.2

6.4 + 0.2 38.8 + 1.1

6.4 + 0.3 6.0 + 0.2 40.4 + 0.9 38.4 + 0.4

3.2 + 0.1 94 + 0.2

3.1 + 0.0 94 + 0.2

3.1 + 0.1 94 + 0.3

2.9 + 0.0 93 + 0.2

78 + 1a 83 + 1a

76 + 1a 81 + 1a

76 + 1a 81 + 1a

69 + 1b 75 + 1b

*W: Acid whey. *WG: Acid whey þ glutamine (15% of total amino acids). *WA: Acid whey þ arginine (9% of total amino acids). *CGW: Casein þ wheat protein hydrolysate þ acid whey (39:39:22). **PER: Protein efficiency ratio (weight gain in g/protein intake in g during the refeeding period). **Digestibility (%): (absorbed nitrogen/ingested nitrogen)6100 D=((I7F)/I)*100. **NPU(%): (retained nitrogen/ingested nitrogen)6100 NPU=((I7 F7U)/I)*100. **BV(%): (retained nitrogen/absorbed nitrogen)6100 BV=(I7 F7U)/(I7F))*100. where I=ingested nitrogen; F=faecal N excretion; U=urine N excretion. Values not sharing the same letter are significantly different (P50.05).

glutamine. When glutamine was supplemented in the diet in the form of protein (group CGW), muscle glutamine concentrations were significantly lower as compared to those of rats fed the acid whey based diet. In the liver, rats fed the CGW diet exhibited similar muscle glutamine levels compared to rats fed the acid whey diet. Interestingly, the addition of arginine to acid whey (at 9% of total amino acids) led to a decrease in the hepatic and skeletal muscle free glutamine concentrations, compared to those observed for animals fed the control diet. Table 6 shows the glutaminase and glutamine synthetase specific activities in jejunum mucosa and liver of rats fed the different experimental diets after 22 days. The hepatic glutamine synthetase activity was significantly higher (P 5 0.05) in rats fed the acid whey based diet compared to rats fed the other experimental diets. Diets did not affect the specific protein content (mg/g tissue) of liver and muscle tibialis. Glutamine or arginine supplementation did not affect skeletal muscle specific protein content. On the other hand, arginine supplementation of acid whey proteins led to a significant increase in liver specific protein content, compared with that of rats fed the control diet. As Table 7 shows, there were differences among the different groups on the jejunum mucosa specific protein content. This was slightly higher in the mucosa of rats fed the WG diet as compared to that of the other groups of animals. Table 5 Plasma amino acid concentrations (mmol/L) of rats fed the experimental diets (mean + SEM, n=8) Amino acid

W*

WG*

Tau 158 + 25a 149 + 20a Asp 17 + 4 14 + 3 Thr 419 + 29 377 + 36 Ser 412 + 19a 415 + 18a Asn 85 + 7 79 + 7 Glu 124 + 9 120 + 11 Gln 1218 + 51a 1190 + 51a Pro 195 + 10 189 + 12 Gly 434 + 18 436 + 24 Ala 434 + 45a 416 + 13a Cit 119 + 5 122 + 7 Val 184 + 12 185 + 10 Cys 27 + 1 26 + 1 Met 62 + 6 60 + 4 Ile 114 + 12 121 + 10 Leu 157 + 5a 173 + 11ab Tyr 107 + 10 91 + 11 Phe 63 + 5 66 + 6 Trp 63 + 6 72 + 5 Orn 76 + 7a 78 + 7a Lys 758 + 49a 780 + 38a His 57 + 3a 57 + 3a Arg 160 + 10 154 + 11 Total 8411 + 510 8194 + 435 Urea 3030 + 155a 2775 + 159a Gln/Urea 0.40 + 0.03a 0.43 + 0.03a Ala/Urea 0.14 + 0.01a 0.15 + 0.02a

WA*

CGW*

150 + 24a 12 + 3 424 + 31 386 + 18a 70 + 8 107 + 10 1030 + 48b 183 + 11 428 + 21 301 + 16b 109 + 8 187 + 8 24 + 3 53 + 3 125 + 10 173 + 12ab 114 + 11 75 + 7 77 + 8 76 + 7a 935 + 34b 51 + 5a 162 + 9 8809 + 505 3509 + 186b 0.29 + 0.03b 0.09 + 0.02b

86 + 10b 16 + 1 463 + 32 524 + 19b 77 + 4 129 + 5 1242 + 46a 201 + 4 479 + 37 379 + 16a 119 + 6 205 + 7 25 + 3 54 + 2 132 + 6 182 + 6b 97 + 7 67 + 2 71 + 3 104 + 7b 923 + 38b 70 + 3b 149 + 5 8982 + 429 3135 + 172ab 0.40 + 0.03a 0.12 + 0.02ab

Values not sharing the same letter are significantly different (P50.05). *W: Acid whey. *WG: Acid whey þ glutamine (15% of total amino acids). *WA: Acid whey þ arginine (9% of total amino acids). *CGW: Casein þ wheat protein hydrolysate þ acid whey (39:39:22).

CLINICAL NUTRITION

323

Table 6 Glutaminase and glutamine synthetase specific activities in the liver and jejunum mucosa of rats fed the different experimental diets (nmol/min/mg protein) (mean + SEM, n=8) Diet W* WG* WA* CGW*

Glutaminase Jejunum mucosa

Glutaminase liver

Gln synthetase liver

Gln synthetase muscle

110 + 11 121 + 4 129 + 5 128 + 7

24 + 2 28 + 2 25 + 2 23 + 2

6.8 + 0.03a 6.0 + 0.2b 5.6 + 0.3b 5.7 + 0.3b

3.6 + 0.2a 2.7 + 0.3b 2.9 + 0.3ab 3.0 + 0.2ab

Values not sharing the same letter are significantly different (P50.05). *W: Acid whey. *WG: Acid whey þ glutamine (15% of total amino acids). *WA: Acid whey þ arginine (9% of total amino acids). *CGW: Casein þ wheat protein hydrolysate þ acid whey (39:39:22).

Table 7 Tissue protein content of rats fed the different experimental diets (mg/g tissue) (mean + SEM, n=8) Diet W* WG* WA* CGW*

Jejunum mucosa

Liver

Muscle tibialis

99 + 4ab 108 + 3a 93 + 2b 91 + 4b

160 + 5a 168 + 6ab 171 + 4b 166 + 5ab

139 + 12 137 + 9 154 + 17 142 + 15

Values not sharing the same letter are significantly different (P50.05). *W: Acid whey. *WG: Acid whey þ glutamine (15% of total amino acids). *WA: Acid whey þ arginine (9% of total amino acids). *CGW: Casein þ wheat protein hydrolysate þ acid whey (39:39:22).

Discussion These studies were designed to examine glutamine and arginine supplementation under conditions of normal growth without significant stress. The supplementation of the protein source (acid whey) with either glutamine or arginine did not lead to any significant change in any parameters concerning body growth and nitrogen balance. In fact, this would not be considered a real supplementation, due to the fact that diets were isonitrogenous; a reduction in essential amino acid contents balanced the increase in glutamine or arginine. However, this fact did not affect rat growth, even though rats were fed 10% protein diets, which could be limiting for some essential amino acids for growing rats. Rats fed the CGW diet exhibited body weight gain, NPU and BV values 10% lower than those fed diets W, WG or WA (P50.05 for NPU and BV values). The major difference in the diets likely responsible is the content of lysine, which is lower in diet containing wheat protein hydrolysate as compared to that of acid whey based diets. Glutamine supplementation of the diet did not lead to an increase in the plasma glutamine levels after 3 weeks of diet consumption. Moreover, the addition of free glutamine to acid whey did not lead to a significant increase in muscle nor liver glutamine. These results are in agreement with those of Hankard et al. (10) found in human volunteers. They observed that oral glutamine administration resulted in a decrease of the de novo synthesis of this amino acid.

Likewise, in vitro, increasing the concentration of glutamine directly inhibits the activity of glutamine synthetase in cultured muscle cells (19). On the other hand, Houdijk et al. (8) have been recently shown that feeding a glutamine-enriched enteral formula increased plasma concentrations of glutamine and arginine in patients with multiple trauma. However, the results of the present study were obtained in healthy growing rats and cannot be compared to those obtained in stressed conditions. As we have stated in the Results section, in rats fed the WG (acid wheyþfree glutamine) the availability of exogenous glutamine also decreased the hepatic glutamine synthetase activity. Such a down-regulation of glutamine synthetase activity makes sound physiological sense. In the muscle tibialis, glutamine synthetase activity was also significantly higher in rats fed the acid whey based diet compared to rats fed the glutamine supplemented diet. The glutaminase activity in the small intestine mucosa of rats fed the W diet tended to be lower compared to the rest of the groups. This would mean that glutaminase activity is substrate dependent, taking into account that diets WG, and CGW had a glutamine content of 15% and, hence, glutamine utilisation (the main energy source for the enterocyte cells and the gut associated lymphoid system) would be higher compared to rats fed diets with a lower glutamine content. Likewise, it has been reported elsewhere that glutamine, itself, provided either orally or intravenously, upregulates the glutaminase activity (20). In the liver, there were no differences between groups and levels remained low compared to those in gut mucosa. It is possible that the small increase in glutaminase activity concluded to be non in rats fed the diet WG, reflected a type II error. Rats fed the diet WA showed higher plasma concentrations of urea and lower concentrations of glutamine compared to those of rats fed the control diet or the control diet plus glutamine. These data agree with the hypothesis that feeding diets poor in arginine (whey proteins) activate the synthesis de novo of glutamine in the liver. It has also been shown that dietary arginine supplementation leads to an increase in the activity of the hepatic urea cycle (21, 22). In this

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GLUTAMINE AND ARGININE ON GLUTAMINE BODY STORES

study, rats fed the arginine supplemented diet exhibited lower plasma glutamine levels and higher urea concentrations than those fed the control diet or the same diet with added glutamine. Haussinger (23) has suggested that the high affinity of glutamine synthetase for ammonia in perivenous hepatocytes allows for the scavenging of ammonia that escapes urea synthesis in periportal cells due to the lack of urea cycle intermediates (mainly, arginine). Re´mesy et al. (24) has recently shown in rats that there are potent mechanisms of nitrogen sparing in the liver involving a shift of ammonia nitrogen utilisation from urea towards glutamine. The protein source of the diet WA, contained 8% arginine (above the total amino acid content), which is high compared to its level in the other diets (2–3%). However, plasma levels of arginine were not different. These experiments would have resolved a difference of 10% statistically significant (P50.05) had it been present. Interestingly, the addition of arginine to acid whey (at 9% of total amino acids) led to a decrease in the hepatic and skeletal muscle free glutamine concentrations, compared to those observed for animals fed the control diet. These results were in agreement with plasma glutamine concentrations and with the observation that arginine supplementation inhibits, somehow, the synthesis de novo of glutamine. In fact, the present study shows evidence that hepatic glutamine synthetase specific activity was lower in rats fed the arginine supplemented diet, compared those of rats fed the control diet. Jejunum mucosa specific protein content was slightly higher in the mucosa of rats fed the WG diet as compared to that of the other groups of animals, even those which received diets containing the same amount of glutamine as diet WG, but as protein (group CGW). The molecular form of the glutamine in the diet affected the rate of not only absorption, but also utilization of this amino acid. Glutamine supplementation of both parenteral and enteral nutrition formulas (as free glutamine) has been associated with improved intestinal mucosal integrity, and improved healing of the mucosa (25). In conclusion, rats fed whey protein (6.4% glutamine) based diets, showed comparable glutamine body stores than rats fed glutamine-supplemented diets (glutamine: 15% of total amino acids). If glutamine content of diets is insufficient to satisfy cellular demands (especially for those tissues with a rapid turnover), de novo synthesis of glutamine from amino acid precursors must be as efficient as exogenous glutamine, in healthy growing rats. On the contrary, arginine supplementation of the diet (up to 9% of the protein content) resulted in a decrease of hepatic and muscle glutamine synthetase activity, that ended up in a parallel decrease in glutamine body stores. Whether the observed effects are valid in situations of stress, remain to be determined.

Acknowledgments Authors would like to thank J.C. Maire for help in the statistical analysis, B. German for helpful discussion and D. Arlettaz for help in analytical work.

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Submission date: 21 October 1999 Accepted date: 18 February 2000

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