Effect of intravenous glutamine on duodenal mucosa protein synthesis in healthy growing dogs

J. Sérgio Marchini1,2, Patrick Nguyen3, Jack-Yves Deschamps3, Pascale Maugère1, Michel Krempf1, and Dominique Darmaun1,4

1 Centre de Recherche en Nutrition Humaine, and 3 Laboratoire de Nutrition et Alimentation, Ecole Nationale Vétérinaire de Nantes, 44093 Nantes, France; 2 Division of Clinical Nutrition, School of Medicine of Ribeirão Preto, 14049-900 Ribeirão Preto, Brazil; and 4 Nemours Children's Clinic, Jacksonville, Florida 32207


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To determine whether glutamine acutely stimulates protein synthesis in the duodenal mucosa, five healthy growing dogs underwent endoscopic biopsies of duodenal mucosa at the end of three 4-h primed, continuous intravenous infusions of L-[1-13C]leucine on three separate days, while receiving intravenous infusion of 1) saline, 2) L-glutamine (800 µmol · kg-1 · h-1), and 3) isonitrogenous amounts of glycine. The three infusions were performed after 24 h of fasting, a week apart from each other and in a randomized order. Glutamine infusion induced a doubling in plasma glutamine level, and glycine caused a >10-fold rise in plasma glycine level. During intravenous infusions of [13C]leucine, the plasma leucine labeling attained a plateau value between 3.22 and 3.68 mole % excess (MPE) and [13C]ketoisocaproate ([13C]KIC) of 2.91-2.84 MPE; there were no significant differences between glutamine, glycine, and saline infusion days. Plasma leucine appearance rate was 354 ± 33 (SE), 414 ± 28, and 351 ± 35 µmol · kg-1 · h-1 (not significant) during glycine, saline, and glutamine infusion, respectively. The fractional synthetic rate (FSR) of duodenal mucosa protein was calculated from the rise in protein-bound [13C]leucine enrichment in the biopsy sample, divided by time and with either plasma [13C]KIC or tissue free [13C]leucine as precursor pool enrichment. Regardless of the precursor pool used in calculations, duodenal protein FSR failed to rise significantly during glutamine infusion (65 ± 11%/day) compared either with saline (84 ± 18%/day) or glycine infusion days (80 ± 15%/day). We conclude that 1) plasma [13C]KIC and tissue free [13C]leucine can be used interchangeably as precursor pools to calculate gut protein FSR; and 2) short intravenous infusion of glutamine does not acutely stimulate duodenal protein synthesis in well-nourished, growing dogs.

stable isotopes; nutrition; small intestine; [13C]leucine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH GLUTAMINE CAN BE SYNTHESIZED de novo and is therefore considered a nonessential amino acid, it is believed to play a significant role in the regulation of protein balance (21). A decade ago, animal studies revealed a unique correlation between free glutamine concentration and the rate of protein synthesis in skeletal muscle (20, 22). This correlation has not, however, been a universal finding: in other animal studies, the size of muscle glutamine pool failed to correlate with the protein synthesis rate (37). In humans, supplementation of intravenous nutrition with glutamine or glutamine-containing dipeptides enhanced nitrogen balance in patients undergoing major gastrointestinal surgery (32, 35) or bone marrow transplantation (39). Glutamine may affect protein balance by enhancing rates of protein synthesis rather than through inhibition of protein breakdown, because we observed in a previous study that a doubling in plasma glutamine concentration, achieved through enteral glutamine infusion, enhanced nonoxidative leucine disposal, an index of whole body protein synthesis, in healthy adult volunteers in the postabsorptive state compared with saline or glycine infusion (15). However, in another study no alterations were observed in leucine and phenylalanine kinetics when healthy adult volunteers were fed a complete amino acid mixture supplemented with oral glutamine (17). The potential role of glutamine as an anabolic agent, therefore, remains to this day controversial.

In addition, the specific organ site of glutamine's putative anabolic effect remains uncertain. Because glutamine is extensively extracted in the small intestine (24, 31), several groups have hypothesized that glutamine may stimulate gut protein synthesis (13, 16, 26, 31). Accordingly, glutamine has a trophic effect on the small intestine of rodents submitted to several models of stress (13, 26, 31). A few studies in humans point to a better preservation of small intestinal structure or absorptive function in critically ill patients receiving glutamine-supplemented parenteral nutrition compared with conventional glutamine-free intravenous nutrition (33, 34).

The aim of this study, therefore, was to determine whether a short-term intravenous infusion of glutamine was able to enhance gut mucosa protein synthesis in healthy fasting dogs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. All studies were conducted in accordance with current guidelines from the French Department of Agriculture for the care and use of animals in biological research. After approval by the National School of Veterinary Medicine at Nantes, five young (142.2 ± 0.1-day-old) beagle dogs participated in the study. Each animal participated in three experiments performed 7 days apart and in a randomized order. Only healthy animals that had a hematocrit >38%, a leukocyte count below 18,000/mm3, a good appetite, normal stools, and body temperature between 38.5 and 39.5°C and were on no medication were studied. The animals' weight (8.28 ± 0.12 kg) did not change between experimental days.

Materials. Natural L-glutamine and glycine were obtained from Sigma (St. Louis, MO). L-[1-13C]leucine (99% 13C) was obtained from MassTrace (Woburn, MA). Tracer solutions were determined to be pyrogen free (limulus lysate assay), sterile (plate culture), and 99% chemically and optically pure before use. Stable isotope tracers were weighed on a high-precision scale and dissolved in known volumes of sterile 0.9% NaCl. Solutions were prepared no earlier than 24 h before study, sterilized by passing through a 0.22-µm filter, stored in sterile sealed containers, and kept at 4°C until used. The concentration of tracer was verified by gas chromatography-mass spectrometry (GC-MS) in triplicate aliquots of each infusate.

Protocol design. Dogs were studied after 24 h of fasting, on three different days, in random order, and with an interval of 1 wk between infusions. On the basal experiment day, each dog received a parenteral infusion of 0.9% NaCl (saline). On another study day, dogs were studied under identical conditions except that they received parenteral glutamine infusion. Finally, each dog underwent an identical study while it received parenteral glycine infusion. To determine whether the rate of incorporation of tracer into mucosal protein was stimulated by glutamine, distal duodenal mucosal biopsies were taken under direct endoscopy at 4 h of tracer and substrate infusion.

Before each study the dogs received standard chow diet for 5-7 days. The diet supplied 9.0 ± 0.3 g · kg-1 · day-1 of protein and 120 ± 3 kcal · kg-1 · day-1. Arterial blood samples were taken from catheters (Davol, Gentilly, France) inserted into the carotid artery 3-4 days before the first experiment to avoid stress. Immediately after its insertion, the catheter was flushed with heparin (10% or 200 U/ml).

On the day of the experiment at 7 AM, two short intravenous catheters were placed into superficial veins of the forelimb, one for infusion of tracer and the other for infusion of saline, glutamine, or glycine. At 7:45 AM, a baseline blood sample was obtained to determine background isotope enrichment in plasma free leucine and alpha -ketoisocaproate (KIC) and protein-bound leucine, and at 8 AM, each dog received a priming dose of L-[1-13C]leucine (approx 10 µmol/kg); the prime was immediately followed by a continuous 4-h intravenous infusion (9.93 ml/h) of a solution of L-[1-13C]leucine (7.98 ± 0.04 µmol/ml) delivered by means of a calibrated Bioblock syringe-pump.

Simultaneously with the tracer infusion, intravenous infusion of saline or unlabeled amino acid was initiated at 8 AM. On the glycine study day, the dogs received an intravenous infusion of 1,783 ± 111 µmol · kg-1 · h-1 glycine (1,783 ± 111 µmol N · kg-1 · h-1) at a rate of 8.1 ± 0.6 ml · kg-1 · h-1. On the glutamine study day, the dogs received 970 ± 135 µmol · kg-1 · h-1 natural L-glutamine (1,942 ± 269 µmol N · kg-1 · h-1) at the rate of 8.6 ± 1.3 ml · kg-1 · h-1.

After the baseline blood sample (time 0), 5 ml of arterial blood were taken in EDTA tubes 180, 200, 220, and 240 min after the start of the isotope infusion. The tubes were kept on ice until centrifugation at 4°C at 2,000 rpm for 10 min. Plasma was immediately separated and frozen at -70°C.

A duodenal biopsy was performed after 4 h of infusion with peroral fibroscopy after a short (20-min) anesthesia with ketamine hydrochlorate. Five or six separate samples from different mucosal sites were pooled to obtain approx 25 mg of tissue and were promptly frozen in liquid nitrogen immediately on sampling and stored at -70°C until analysis.

Analytic methods. Plasma amino acid concentrations were determined on an amino acid analyzer (Beckman, Anaheim, CA).

Total plasma protein from the preinfusion samples, precipitated by use of 12% (wt/vol) trichloroacetic acid (TCA), was used to estimate background body protein [13C]leucine labeling for use in the calculation of mucosal protein synthetic rate. In our experience, the background 13C enrichment of plasma free leucine and protein-bound leucine was consistently about -28 to -27per thousand (expressed as delta per thousand vs. Pee Dee Belemnite).

To isolate amino acids in tissue and plasma, frozen tissue samples were homogenized in 2 ml of ice-cold 12% TCA and centrifuged at 2,000 g for 20 min. The supernatant containing tissue fluid free amino acids was removed and dried under nitrogen. After three washes with 12% TCA, the protein pellet was hydrolyzed in 6 M HCl at 110°C for 24 h and then dried. Total plasma proteins were isolated in a similar manner. In preliminary experiments, the initial biopsy sample was spiked with [2H3]leucine; we verified that the final sample obtained from hydrolyzed pellet protein did not contain any [2H3]leucine, attesting that there was no significant "contamination" of pellet protein by supernatant free amino acid.

Leucine in all preparations was isolated by cation exchange chromatography and derivatized to its N-acetyl, n-propyl ester derivatives. The isotopic enrichment of leucine was determined in plasma and mucosal supernatant using methane chemical ionization GC-MS on a 5890 series II gas chromatograph coupled with a 5971A mass selective detector (Hewlett-Packard) equipped with an ultra-1 capillary column (25 m × 0.32 mm ID, 0.17 mm film thickness; Hewlett-Packard). The temperature of the injection port was 250°C, and the oven temperature was initially set at 80°C and then ramped at 15°C/min up to 160°C and then at 40°C/min up to 250°C. Selected ion monitoring was used on ions of mass-to-charge ratio (m/z) 216 and 217, representing the molecular ions of natural and 13C-labeled leucine, respectively. When the observed 217/216 ion current ratios were plotted as a function of the expected 13C enrichment to natural mole ratio, linear regression coefficients >0.98 were consistently observed, along with a slope >0.94 for the standard curve.

Low isotopic enrichments in leucine derived from the hydrolysis of pellet protein or total plasma protein were determined by GC-combustion-isotope ratio MS (known as GC-C-IRMS; 5890 series II GC, Hewlett-Packard and Delta S IRMS, Finnigan MAT) equipped with an ultra-1 capillary column (25 m × 0.32 mm ID, 0.17 mm film thickness; Hewlett-Packard), as previously described (38). The temperature of the injection port was 250°C, and the temperature program was initially set at 80°C and then ramped at 8°C/min up to 160°C and then at 40°C/min up to 230°C for 2.5 min. When the observed ratios were plotted as a function of the expected 13C enrichment to natural mole ratio, linear regression coefficients >0.98 were observed, along with slopes >= 0.92 for the standard curve.

13C enrichment in plasma alpha -ketoisocaproic acid (KIC) was used as an index of intracellular leucine enrichment. Plasma KIC was isolated from plasma and converted to its oxime-tert-butyldimethylsilyl derivative, as previously described (9). Isotopic enrichment was measured by electron impact ionization GC-MS by monitoring ions at m/z 316 and 317 by use of a DB-1 capillary column (30 m × 0.25 mm ID, 0.25 mm film thickness, J&W Scientific) operated in the splitless mode. The temperature of the injection port was 250°C, and the temperature program was initially set at 80°C and then ramped at 10°C/min up to 220°C. When the observed 317/316 ion current ratios were plotted as a function of the expected 13C enrichment to natural mole ratio, linear regression coefficients >0.98 were consistently observed, along with a slope >0.98 for the standard curve.

Calculations. Isotopic steady state in plasma leucine and KIC was established by demonstrating with linear regression that the slope of the isotope enrichment against time was not different from zero.

Total leucine flux (µmol · kg-1 · h-1) was calculated as
Flux = i[(E<SUB>i</SUB> /E<SUB>p KIC</SUB>) − 1]
where i is the tracer infusion rate (µmol · kg-1 · h-1), Ei and Ep KIC are the isotopic enrichments in the infused leucine and in plasma KIC at plateau, respectively.

Fractional synthesis rate (FSR), corrected for 24 h, of total duodenal mucosa protein was calculated according to the precursor/product relationship
FSR = (E<SUB><IT>t</IT>,prot</SUB> − E<SUB>0,prot</SUB>) / (E<SUB>precusor</SUB> × <IT>t</IT>)
where E0,prot and Et,prot are the 13C enrichments in the bound leucine residues in total plasma protein at time 0 and in duodenal protein at time t, respectively. Eprecursor is the 13C enrichment of the precursor (plasma Leu or KIC or tissue fluid free leucine), and t is the time of the tissue biopsy (time 0 being the time of beginning of the isotope infusion).

Statistical analysis. Data are presented as means ± SE. Data were compared between treatments by repeated-measures ANOVA, a paired Student's t-test, and Kruskall-Wallis nonparametric tests. Significance was established at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasma amino acid concentrations (Table 1) were measured twice during the last 20 min of each glutamine, glycine, or saline infusion, when a steady state was apparent on the basis of [13C]leucine plateau (see below). Glutamine infusion induced a doubling in plasma glutamine level, and glycine caused a >10-fold rise in plasma glycine level. In contrast, neither glutamine nor glycine infusion altered the plasma concentrations of leucine, KIC, or of most other essential or nonessential amino acids.

                              
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Table 1.   Plasma amino acid concentrations after 4 h of glutamine, glycine or saline infusion

During intravenous infusion of [13C]leucine, the plasma leucine labeling attained a plateau value between 3.22 and 3.68 mole % excess (MPE), and [13C]KIC of 2.91-2.84 MPE, and there were no statistical differences during infusion of glutamine, glycine, or saline. There were no differences for leucine flux calculated from plasma KIC enrichment (Table 2).

                              
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Table 2.   Effect of intravenous glutamine and glycine infusion on plasma KIC and leucine kinetic parameters in 5 growing dogs

FSRs of duodenal mucosa protein were calculated from different precursor pool enrichments (Table 3). The results obtained from tissue fluid free leucine pool and plasma KIC were not significantly different, and they were higher than those calculated using the plasma [13C]leucine pool. Again there were no statistically significant differences after glutamine or glycine infusion.

                              
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Table 3.   Effect of glutamine and glycine infusion on FSR of duodenal mucosa protein calculated with different precursor pools in 5 young dogs


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study used continuous infusion of stable isotope-labeled leucine to examine the effect of a short, intravenous infusion of natural glutamine on the FSR of duodenal mucosa protein in healthy, growing dogs. We observed that 1) the use of either plasma KIC or tissue fluid free leucine as a precursor pool resulted in estimates of protein FSR that were not statistically different; and 2) regardless of the precursor pool used for calculations, glutamine failed to stimulate duodenal protein FSR compared with either saline or isonitrogenous glycine infusion.

The lack of stimulation of gut protein FSR by glutamine observed in the current study is at odds with several previous studies documenting stimulation of cell proliferation by glutamine in human or rat ileum in vitro (16, 26, 29) or enhanced protein content in rat jejunum (13) and prompts us to question either the isotopic approach used, the dose of glutamine, the animal model, or the study design used.

In vivo assessment of the rates of tissue protein synthesis has relied either on continuous tracer, most commonly [13C]leucine, infusion or on the flooding dose method (14, 28, 30). Although each of these approaches has its own limitations, the main limitation to the continuous infusion method is the inability to directly assess the isotopic enrichment of the direct precursor amino acid pool used for tissue protein synthesis, i.e., tRNA [13C]leucine. Previous studies have suggested that plasma KIC could be used as the precursor pool in lieu of tissue free leucine in muscle and liver (2, 4). The current study documents that isotopic enrichments did not differ significantly between plasma KIC and gut free leucine. The intersubject coefficient of variation (CV = 100 × SD/mean) in measured enrichments was, however, approx 30% in the saline infusion experiments (with an SD of 0.89 for a mean of 2.89 MPE; Table 3). It can thus be estimated that, in a paired study design, nine dogs would be required to detect a 30% difference in measured enrichments between KIC and mucosal free leucine with an alpha  <0.05 and 1 - beta  = 0.80. With this reservation in mind, the data nevertheless suggest that KIC may be used as a fair estimate of the precursor pool for protein synthesis in the gut, even during intravenous administration of large amounts of amino acids. Our data are consistent with earlier studies showing that the 13C enrichment of leucyl-tRNA in stomach mucosa was the same as that of plasma KIC during a continuous infusion of [13C]leucine in pigs (8).

For this reason, it is unlikely that inaccurate assessment of precursor pool enrichment accounts for the apparent lack of effect of glutamine. Similarly, the intravenous route of tracer infusion is unlikely to affect the results, because other workers have reported that similar gut protein FSR could be obtained by use of either intravenous or enteral [13C]leucine (25).

We used the [13C]leucine enrichment in mixed plasma protein-bound leucine as an estimate of the baseline enrichment in gut protein-bound leucine. The choice is reasonable for the first isotope infusion day, since there is no reason to believe that, in "naive" dogs that have never received any tracer infusion, [13C]leucine content should differ between tissues, yet this may not apply for the second and third infusion days. Because the bulk of mixed plasma protein is albumin, which has a turnover rate of only approx 8-10% per day (11), the residual [13C]leucine enrichment in plasma albumin may largely exceed that in gut protein, which turns over at a rate of approx 60-90% per day. To address this issue, additional experiments were carried out (data not shown) in which gut protein-bound [13C]leucine enrichment was measured 1 wk after the performance of a [13C]leucine infusion in a single dog. Residual enrichment remained at 0.0440 MPE in plasma protein vs. only 0.0234 MPE in gut protein in samples obtained simultaneously. The use of same-day plasma protein enrichment as a baseline thus overestimates the residual enrichment in gut protein and, as a consequence, underestimates the rate of [13C]leucine incorporation and gut protein FSR during the second and third infusion days. On the other hand, using the first-day initial value of plasma protein enrichment before the first isotope infusion will result in underestimating residual enrichment and overestimating gut protein synthesis during the second and third isotope infusions. Table 4 displays results from two sets of calculations that use either same-day or first-day plasma protein [13C]leucine content as an estimate of baseline 13C abundance in gut protein. Although the absolute values of gut protein FSR were altered depending on the baseline chosen, we failed to detect any significant rise in gut protein FSR on the glutamine infusion day.

                              
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Table 4.   Gut protein FSR calculated with tissue free leucine or plasma KIC as precursor pool: effect of using either same-day plasma protein or first-day plasma protein as an estimate of baseline residual [13C]leucine enrichment in gut protein

On the other hand, the putative protein anabolic effect of glutamine may be species specific, and it may be nonexistent in the dog. The dog has been used extensively as a model for the investigation of whole body leucine metabolism (1, 2). Moreover, because glutamine is extensively taken up by the gut and inhibits lipolysis and ketogenesis in dogs (5, 6), as it does in humans (10), this animal model seemed reasonably similar to humans with regard to glutamine metabolism. Whereas earlier studies documenting intestinal glutamine extraction, as well as glutamine's inhibitory effect on lipolysis, used 96-h-fasted adult dogs (5, 6), we used young, growing dogs after a 24-h fast. In addition, among the many metabolic effects attributed to glutamine, inhibition of leucine oxidation and enhancement of nonoxidative leucine disposal, together an index of whole body protein synthesis, have only been documented in healthy humans (15), not in healthy dogs; accordingly, whereas glutamine infusion decreased plasma leucine concentration in studies performed in humans (10, 15), glutamine failed to affect plasma leucine level in the current report.

The lack of effect of glutamine may be due to the experimental design itself, e.g., a type II statistical error, the fasting condition of the animals, their preexisting nutritional status, or the dosage or timing of glutamine infusion.

In theory, the small size of the population studied could have precluded the detection of glutamine's anabolic effect: indeed, given the large intersubject CV (= 100 × SD/mean) in gut protein FSR (e.g., approx 48% in the saline infusion experiments), the minimal change that could be detected with 80% power and an alpha  <0.05 would be an approx 67% change in gut FSR (when a 2-sided test is used) or an approx 55% rise (when a 1-sided test is used) (7). Whereas the purpose of the study was to determine whether glutamine was able to enhance gut protein FSR, there was, however, a trend (not significant) toward a lower (rather than an enhanced) rate of gut protein FSR on the glutamine infusion day (Table 3) compared with saline or glycine infusion.

Glutamine was administered to animals in the fasting state, and the gut may only be capable of mounting a protein anabolic response in the fed state. However, in previous studies, glutamine enhanced whole body estimates of protein synthesis in postabsoptive humans (15). The amounts of glutamine infused into our dogs (970 µmol · kg-1 · h-1, i.e., 0.57 g/kg over 4 h), were equivalent to 100% (39) and 200% (19) of the daily doses administered by others to critically ill humans: insufficient dosage therefore hardly seems a plausible explanation for the lack of effect of glutamine. However, in rat skeletal muscle, protein synthesis may correlate with the concentration of free glutamine (20). If the same is true for the small intestine, the failure of glutamine to elicit an anabolic effect in the current study may reflect an inability to increase intracellular glutamine concentration; although plasma glutamine doubled, glutamine concentration was not measured in the duodenal mucosa in the current study.

The duration of the glutamine supplement may have been too short to elicit any anabolic effect. Accordingly, even though a 5-day glutamine supplementation enhanced both nitrogen balance and polyribosome concentration in skeletal muscle from surgical patients (35), a short, 5.5-h course of intravenous glutamine failed to affect muscle protein synthesis after surgery (19). Also, preexisting depletion of the body glutamine pools may be a prerequisite for glutamine supplementation to elicit its protein anabolic effect. Although the size of muscle or gut glutamine pools was not assessed in the current studies, we speculate that glutamine pools were intact at baseline in the animals studied, because they had received ample amounts of protein, and therefore large amounts of glutamine as a protein-bound residue, in their diet before the study. Earlier studies indeed suggest that rates of protein synthesis in the gut may depend on dietary protein intake in growing animals (37).

It should be pointed out that the lack of stimulation of mixed duodenal protein synthesis by acute glutamine infusion does not rule out a beneficial effect of glutamine on small intestinal mucosa. Indeed, glutamine may enhance the synthetic rate of a few specific proteins in the tissue without altering the overall FSR of mixed gut protein. In addition, a trophic effect of glutamine could be mediated through a number of other mechanisms, including 1) a simple "nutritional" effect, as glutamine provides energy to gut mucosa (21, 24, 31); 2) increased synthesis of DNA, because glutamine is a known donor of nitrogen for the synthesis of purines and pyrimidines (12), the building blocks of nucleic acids that are in high demand during cell replication; 3) enhancement of intestinal immune function, as glutamine is the main fuel for lymphocytes and macrophages (3); 4) increased intestinal blood flow (18); or 5) enhanced glutathione synthesis, because intestinal glutathione plays a critical role in the defense of mucosal cells against oxidative stress (23), and glutamine may be a source for glutathione synthesis in the gut (27).

Finally, the organ site of glutamine's putative protein anabolic effect may be skeletal muscle, rather than the small intestine. Further studies performed in dogs undergoing stress and/or protein wasting may be warranted to determine the "target" organ of glutamine's effect on tissue protein homeostasis.


    ACKNOWLEDGEMENTS

We are indebted to Drs. Lucille Martin, Etienne Pouteau, and Henri Dumon for assistance with the handling and surgical preparation of the animals, and to Lynda Everlyne (Biochemical Genetics Lab, Nemours Children's Clinic) for the amino acid concentration assay.


    FOOTNOTES

This study was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-51477 (to D. Darmaun) and grants from the Fondation pour la Recherche Médicale (Paris, France) and Fundação de Amparo à Pesquisa do Estado de São Paulo, Brasil (95/09776-1 to J. S. Marchini).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: D. Darmaun, Centre de Recherche en Nutrition Humaine, CHU Hotel-Dieu, 44093 Nantes cedex 1, France (E-mail: ddarmaun{at}nantes.inserm.fr).

Received 15 January 1998; accepted in final form 10 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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