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© 2007 American Society for Nutrition J. Nutr. 137:1931-1937, August 2007

Nutrition and Disease

Dietary Glutamine Affects Mucosal Functions in Rats with Mild DSS-Induced Colitis1–3,

María Vicario4, Concepció Amat4, Montserrat Rivero5, Miquel Moretó4 and Carme Pelegrí4,*

4 Departament de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain and 5 Laboratorios Ordesa SL, 08830 Sant Boi de Llobregat, Spain

* To whom correspondence should be addressed. E-mail: carmepelegri{at}ub.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The development of inflammatory bowel disease may involve immune dysfunction. Because enteral glutamine is the main source of amino acids for the intestinal mucosa and is metabolized at high rates by both enterocytes and immunocytes, the aim of this study was to ascertain the protective role of glutamine supplementation in a DSS-induced model of mild experimental colitis on metabolic, immune, and intestinal variables. Lewis rats were fed diets supplemented with glutamine (glutamine diet, G group) or an isoenergetic isonitrogenous control diet (C group) from postnatal d 21 (weaning) and continuing to d 35. On d 30, half of the rats from both groups were given 0.5% DSS in drinking water (G-DSS and C-DSS groups). Glutamine supplementation increased the plasma concentrations of Thr, Gln, Cit, His, and Arg and enhanced the ratio of essential to nonessential amino acids irrespective of DSS treatment. DSS administration increased the plasma Gln concentration, indicating a reduced utilization of this amino acid by the intestinal tissue. Regarding the gut-associated lymphoid tissue lymphocyte populations, DSS increased the percentages of CD3+ T lymphocytes from Peyer's patches, NK and B lymphocytes from mesenteric lymph nodes, and NK CD8– cells from intraepithelial lymphocytes. The administration of glutamine did not affect the inductive populations nor did it modify T-cell subtypes or the percentage of intraepithelial lymphocytes of gut-associated lymphoid tissue. However, glutamine supplementation reduced the feces water contents in the DSS-treated but not in the untreated rats. These results indicate that glutamine supplementation can improve barrier function in rats with colitis.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The inflammatory bowel disease, which includes Crohn's disease and ulcerative colitis (UC),6 is a multifactorial chronic disorder of unknown etiology characterized by acute exacerbations followed by remissions. Although several factors are reportedly involved in the development of the disease, including immune dysfunction, genetic susceptibility, and bacterial flora within the intestinal environment, the underlying pathogenic mechanism has not yet been identified (1). Food is also a major factor affecting the intestinal environment and diet has therefore been implicated in its pathogenesis (2). Nutrition has both supportive and therapeutic roles in the management of chronic gastrointestinal diseases. Specific nutrients and/or unbalanced diets can induce disease, whereas dietary factors such as fiber, short-chain fatty acids, fish-oils, or glutamine may prevent gastrointestinal disease (3).

Administration of dextran sulfate sodium (DSS) in the drinking water of rodents is widely used as an experimental model of UC because it results in acute and chronic colitis and shares clinical and histopathological characteristics with human UC (4,5). DSS is a sulfated polymer that induces patchy mucosal injury in a dose-dependent manner (6,7). It also induces inflammation, initially through a direct toxic effect on epithelial cells, with subsequent activation of macrophages, recruitment and activation of inflammatory cells, and upregulation of inflammatory mediators, thereby leading to the development of severe colitis (4,8).

The breakdown of the intestinal barrier defense was shown to increase invasion by antigens, leading to continuous immune system activation (9,10). Because a defect in barrier function is thought to be important in the pathogenesis and systemic manifestations of UC, improvement in mucosal integrity may contribute to a reduction in intestinal inflammation.

Glutamine is the primary source of amino acids for the intestinal mucosa (11). It is the main respiratory substrate for enterocytes and is also important for intestinal metabolism and immune cells, especially in animals exposed to stress (12,13). Glutamine-fortified parenteral and enteral diets significantly improve intestinal morphology and function (14) and, in animal models of colitis glutamine-supplemented diets, resulted in less severe intestinal damage, reduced weight loss and bacterial translocation, and improved nitrogen balance (15–17).

We previously reported that oral administration of low doses of DSS (0.5%) to young rats increases the number of mucin-secreting cells and also induces mucosal hyperemia and mucosal infiltration of neutrophils in the colon already at d 5 of treatment (7), indicating a dysfunction of the mucosal barrier (18). Because the described experimental protocol modifies neither feeding behavior nor growth, it may be a suitable model for studying the effects of dietary supplementation of certain nutrients, such as glutamine, on the course of intestinal barrier alterations during the induction phase of DSS colitis in young rats.

Most of the studies addressing DSS-induced experimental colitis focus on elucidating the immune mechanisms responsible for mucosal damage, which involve immune cell characterization and cytokine profile. However, the gut-associated lymphoid tissue not only encompasses leukocytes in the lamina propria, but also the intraepithelial lymphocytes (IEL) and lymphoid cells in mesenteric lymph nodes (MLN), Peyer's patches (PP), and isolated lymphoid follicles (19). Because different sites of the intestinal immune system interact closely, we sought to characterize the lymphoid population present in the MLN, PP, and IEL following DSS administration. Therefore, our study was designed to assess the implications of innate and acquired intestinal and systemic immunity in a DSS model of mild intestinal inflammation in young rats. A second objective of the study was to test whether prophylactic glutamine supplementation of their diet modifies the physiopathological findings observed during DSS administration.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Rats and experimental design. Litters of male Lewis rats (6 rats/mother), 15 d of age, were purchased from Harlan Ibérica and housed under standard conditions, with a controlled 12-h light:12-h dark cycle. At the day of weaning (21 d of age), pups were randomized (3/cage) to receive either a glutamine-supplemented solid diet (glutamine diet, G group) or an isoenergetic isonitrogenous control diet (C group) for 14 d. The glutamine-enriched diet was based on AIN93G (20) by substituting cornstarch for L-glutamine powder at 50 g/kg diet before pelleting (Dyets). In the control diet, glutamine was replaced by adding a mixture of nonessential amino acids (in g/kg: L-Asp, 13.2; L-Ser, 9.6; L-Gly, 15.6; L-Pro, 10.4; L-Ala, 8.0) to balance the nitrogen and energy content of the glutamine-supplemented diet, following the criteria used by Houdijk et al. (21). After 9 d of feeding the diet, rats (then 30 d of age) from C or G groups were randomly divided into 2 experimental groups: one received DSS in the drinking water and the other received only tap water. Thus, the experimental groups were distributed as follows: control diet (C group); control diet plus DSS (C-DSS group); glutamine diet (G group); and glutamine diet plus DSS (G-DSS group). During the experimental protocol, all rats had free access to food and water. Body weight and food intake were monitored daily from the day of weaning until d 35. After a total feeding period of 14 d (9 d with the C or G diet, followed by 5 d with the specific diet with or without DSS treatment), rats were killed by anaesthesia with isofluorane, followed by cervical dislocation (35 d of age), exsanguinated, and samples of blood, jejunum, and colon were obtained and processed for further analyses. Studies were performed in accordance with the institutional guidelines for the care and use of laboratory animals established by the Ethical Committee for Animal Experimentation at the University of Barcelona.

    Induction of colitis. In the C-DSS and the G-DSS groups, colitis was induced by replacing normal drinking water with water containing 5 g/L DSS (MW = 40 kDa; ICN Biomedicals) for 5 d, as previously described (7).

    Water content in feces. From d 1 to d 5 of DSS treatment (i.e., 31–35 d of age) spontaneously defecated feces from each animal were collected and immediately weighed and dried at 60°C for 2 d to estimate feces water contents during the 5-d treatment. For each rat, the mean value of the measures obtained in the 5-d period was considered, so n reflects independent observations.

    Myeloperoxidase activity. The presence of neutrophils in the intestinal mucosa was estimated by measuring the activity of myeloperoxidase (MPO) in mucosal scrapings from jejunum and colon. Samples were obtained and MPO was extracted and quantified following the protocol described by Vicario et al. (7).

    Blood determinations. Blood samples were obtained by cardiac puncture and collected in EDTA-coated Multivette tubes (Sarstedt). Leukocyte and erythrocyte concentrations, hematocrit, hemoglobin concentration, and mean corpuscular hemoglobin were analyzed in a Coulter Counter JT hemocytometer. To obtain serum, blood was collected in sterile tubes and left for 1 h at room temperature to allow complete clot formation. Samples were centrifuged at 2000 x g for 10 min at 4°C and supernatants were collected and kept at –80°C until quantification of IgA and IgG by ELISA technique and glucose with the Accutrend Sensor (Roche Diagnostics).

    Plasma free amino acid concentration. Blood samples were immediately centrifuged at 2000 x g for 10 min at 4°C to obtain plasma. After the addition of the internal standard (Norleucin, 0.25 mmol/L; Sigma), plasma samples (100 µL) were then deproteinized in trifluoroacetic acid (10%), centrifuged at 17000 x g at 4°C for 30 min, and the supernatants purified by centrifugation through an Ultrafree-MC 10000NMWL membrane filter (Millipore) at 13000 x g for 30 min at 4°C. Quantitative analysis of plasma amino acids was performed by ion-exchange chromatography following the method described by Moore et al. (22). The analyzer (Amino Acid Analyzer Amersham Pharmacia LKB Biotech-Biochrom, model Alpha Plus) was equipped with a cation exchange column (sulphonate polystyrene divinyl-benzene resin, 5 µm particle size; 200 x 4 mm in length and internal diameter, respectively; Biochrom). Chromatographics runs were made using the lithium citrate buffer gradient (pH 2.2) and temperature gradient recommended by the manufacturer for physiological fluids. To avoid glutamine degradation, samples were kept at 4°C before injection. Amino acid concentrations (µmol/L) were calculated from individual peak areas and external and internal standard areas.

    Immunoglobulin quantification. Luminal IgA content was determined in jejunal washings. The small intestine was excised 5 cm from the stomach and divided into 2 parts: the proximal part was considered the jejunum and the distal part the ileum. The luminal content was collected separately by flushing each specimen with 10 mL of ice-cold PBS. The samples were vortexed to homogenize the luminal content and centrifuged at 2000 x g for 10 min at 4°C. The supernatants were collected; snap frozen, and maintained at –80°C until later processing. Serum samples were assayed for IgA and IgG quantification. Immunoglobulin determination was carried out by performing the ELISA with the corresponding capture antibody for IgA (mouse anti-rat IgA, clone MARA-1, 1.25 mg/L; Labgen) or IgG (mouse anti-rat IgG, clone MM711, 0.25 mg/L; Labgen). The amount of Ig in samples was determined through the detection antibody (anti-mouse Ig, clone MARK-1/MARK-15 {kappa}/{lambda}; 0.1 mg/L; Labgen) conjugated to horseradish peroxidase.

    Histology. Intestinal fragments from the distal colon were fixed, dehydrated in graded ethanol, and embedded in paraffin. Sections of 10–12 µm were processed for hematoxylin and eosin staining and tissue architecture and goblet cellularity in the colon were analyzed using a blind protocol.

    Isolation of lymphocyte populations. MLN and PP were excised and samples were prepared as previously described (23). IEL were isolated from the small intestine following the protocol described by Cerf-Bensussan et al. (24) with some modifications (25). Briefly, that part of the small intestine free of PP was cut into fragments of 5 mm and incubated in Ca2+/Mg2+-free Hank's balanced salt solution (Gibco) supplemented with 20% (v:v) FCS, 1 mmol/L Dithiothreitol (Sigma) and antibiotics for 20 min at 37°C with continuous agitation. Fragments were then incubated in RPMI with 10% (v:v) FCS and antibiotics for 40 min at 37°C. The debris were removed by passing the suspension through a nylon wool and thereafter purified with a discontinuous Percoll (Pharmacia) gradient, by resuspending cells in 44% Percoll and centrifuging them (600 x g, for 20 min at 20°C) over 67.5% Percoll. After cells were washed with PBS-FCS-sodium azide, cell count and viability were determined.

    Immunofluorescence staining and flow-cytometric analysis. Isolated cells (2–3 x 105) were stained using a double immunofluorescence technique, as described previously (23). The anti-rat monoclonal antibodies (MAb) used to detect different subsets of lymphocytes were as follows: anti-CD45 (common leukocyte antigen, fluorescein isothiocyanate FITC-conjugated, Pharmingen) for total lymphocytes; anti-CD45RA (OX33, unconjugated; Pharmingen) for B cells; anti-CD5 (OX19, FITC-conjugated; Labgen) for T cells; anti-CD3 (1F4, FITC-conjugated or unconjugated; Pharmingen) for T cells; anti-CD4, (W3/25, FITC-conjugated or unconjugated; Labgen) for T helper lymphocytes; anti-CD8{alpha} (OX-8, FITC-conjugated or unconjugated; Labgen) for T suppressor/cytotoxic lymphocytes; anti-TCR{alpha}ß (R73, unconjugated; Pharmingen) and anti-TCR{gamma}{delta} (V65, unconjugated; Pharmingen) for the type of T cell receptor; anti-CD25 (NDS61, unconjugated; Labgen) for expression of IL-2 receptor; anti-NKR-P1A (10/78, unconjugated, Pharmingen) for natural killer (NK) lymphocytes, and anti-rat Ig FITC-conjugated (Dako Cytomation) for surface expression of immunoglobulins. Anti-mouse IgG (H+L) phycoerythrin PE-conjugated (Sigma) was used as the secondary Ab. All Ab were used at saturating concentrations and mouse anti-human CD7 (124 1D1; kindly provided by Dr. Vilella, Hospital Clínic i Provincial) was used as a negative control staining. Flow cytometry was performed using a FACS Epics Elite (Coulter).

    Statistical analysis. Samples that were analyzed in duplicate or triplicate were treated as single values using the arithmetic mean of replicates. Results are expressed as the mean ± SEM, n = 8–16 rats. Statistical analysis was performed by a 2-way ANOVA using Statistica 6.0 program software (Stat Soft). For each dependent variable studied, the independent factors considered were glutamine administration and DSS treatment. When interaction between both variables was detected, post hoc comparisons (LSD test) were performed. Differences were considered significant at P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Food intake and body weight. Food intake and body weight were measured daily. Neither of these variables nor growth rate was affected by diet or DSS treatment (data not shown). In the glutamine-supplemented groups the calculated glutamine intake was 5.5 g/(kg · d).

    Water content in feces. Oral administration of 5 g/L DSS for 5 d (31–35 d of age) markedly increased the percentage of water in feces (P = 0.001; Fig. 1). Administration of the glutamine diet before and during DSS-treatment partially prevented this effect because the mean water content in feces was lower in the G-DSS group than in C-DSS group (P = 0.0004). No differences were observed between the C and G groups.


Figure 1
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  		FIGURE 1  Water content in feces obtained for 5 d from rats aged 31–35 d fed the control diet (C) or the glutamine diet (G) and untreated or treated with 5 g/L DSS (DSS). Results are expressed as the mean of the 5-d treatment ± SEM, n = 10–12 rats. Means without a common letter differ, P < 0.05.

 
    MPO activity. The 5 d DSS treatment significantly increased the mucosal MPO activity in both the jejunum and the colon (Table 1) without any effects of glutamine supplementation.


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  		TABLE 1 MPO activity in the intestinal mucosa of 35-d–old rats fed the control diet (C) or the glutamine diet (G) and untreated or treated with 5 g/L DSS (DSS)1

 
    Hematological variables. Analysis of hematological variables showed that there were no differences among the groups. Hence, the following values correspond to the overall mean and SEM of the 4 experimental groups (n = 36–48): leukocyte count, 5.22 ± 0.17 x109/L; erythrocyte concentration, 6.49 ± 0.03 x1012/L; hematocrit, 0.380 ± 0.002; hemoglobin, 126 ± 0.053 g/L; mean corpuscular hemoglobin, 19.9 ± 0.06 pg. The concentration of glucose at 35 d of age was 6.33 ± 0.16 mmol/L (n = 40).

    Plasma free amino-acid concentrations. Glutamine supplementation for 15 d increased the plasma Thr, Gln, Cit, His, and Arg concentrations. The G and G-DSS groups had lower plasma Ser, Asn, Pro, and Gly concentrations, which can be explained by the reduced contents of these amino acids in the G diet compared with the C diet (Table 2). DSS lowered the Ile and Glu concentrations and raised that of Gln. Plasma total amino acid and essential amino acid concentrations were not affected by diet or DSS treatment.


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  		TABLE 2 Plasma free amino acid concentrations of 35-d–old rats fed the control diet (C) or the glutamine diet (G) and untreated or treated with 5 g/L DSS (DSS)1

 
    Quantification of IgA and IgG. In all groups, the amount of secretory IgA detected in the intestinal lumen was higher in the distal region (16.12 ± 0.905, n = 32) than in the proximal region of the intestine (9.21 ± 0.705, n = 34) (P < 0.001). Glutamine supplementation or DSS treatment had no effect among any of the groups. The serum concentration of IgA (18.3 ± 2.0 mg/L; n = 12) was unaffected by the glutamine diet or DSS treatment. However, groups treated with oral DSS showed significantly higher concentrations of IgG than untreated rats (with DSS treatment, 560 ± 18 mg/L, n = 30; without DSS, 481 ± 21 mg/L, n = 77; P = 0.0167). Diets supplemented with glutamine did not induce significant changes in this variable.

    Histology. Macroscopic observation of the colonic mucosa did not reveal signs of injury or ulcerations, although some specimens from DSS-treated rats exhibited some hyperemia. Microscopic examination of transmural segments showed that all rats under DSS treatment had higher densities of goblet cells (Fig. 2). Glutamine supplementation did not modify these results.


Figure 2
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  		FIGURE 2  Representative photomicrographs of intestinal sections from distal colon from rats fed the control diet (C) or the glutamine diet (G) and untreated or treated with 0.5% DSS (DSS). Bar = 100 µm.

 
    Phenotype of lymphocytes. Typification of main lymphocyte subpopulations at inductive sites revealed significant changes in the percentages of different cell populations resulting from DSS oral treatment (Table 3). In the PP of DSS-treated rats, there were higher percentages of CD3+ T cells coexpressing the CD5+TCR{alpha}ß+ phenotype. This increase was in accordance with higher percentages of CD4+CD8– lymphocytes, and lead to elevated CD4:CD8 and T:B ratios in DSS-treated rats. However, MLN did not show significant variations in lymphocyte subtypes due to DSS; rather, only a slight increment in B cells (CD45+Ig+) and in the NK lymphocyte subset (P = 0.07) were observed (data not shown). There were no differences in the percentage of activated T cells (CD25+), and the proportion of CD4+ and CD8+ T cells were similar in all groups. Consequently, the CD4:CD8 and T:B ratios did not change significantly between the experimental groups. The addition of glutamine to the diet did not have any effect on these variables.


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  		TABLE 3 Percentages of lymphocyte subpopulations of Peyer's patches of 35-d–old rats fed the control diet (C) or the glutamine diet (G) and untreated or treated with 5 g/L DSS (DSS)1

 
With respect to IELs, CD45RA+Ig+ cells (B lymphocytes) accounted for <2% of total isolated cells, indicating a low contamination of lamina propria leukocytes in the IEL cell suspensions obtained. Staining with CD45 revealed that 97.2 ± 1.2% of IEL-gated cells expressed this surface antigen (data not shown). In all groups, the T-cell subtypes studied showed similar percentage values. However, DSS treatment induced a significant increase in the NK cell populations specifically enhancing the relative amount of NKR-P1A+CD8– (28.03% ± 0.67, n = 29 vs. 23.94% ± 1.02, n = 17, P < 0.003) and not substantially modifying the percentage of NKR-P1A+CD8+ cells. Supplementation of the diet with Gln did not affect IEL percentages (not shown).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The preventive effects of diets supplemented with glutamine on intestinal integrity during experimental severe colitis have been reported by various authors (15–17,26,27). However, there is no information on the effects of dietary glutamine supplementation during the induction of mild intestinal inflammation. Thus, we considered it relevant to further characterize a recently described model of mild intestinal inflammation in young rats based on the use of low doses of DSS (7), and also to determine whether dietary glutamine could attenuate or prevent the development of symptoms of the experimental disease.

The model of ulcerative colitis based on the administration of DSS is well known. Although the exact mechanism of action underlying this agent is not fully understood, it has been shown that the first event occurring in the colonic mucosa is the breakdown of the epithelial mucosal barrier (18). We demonstrated that DSS treatment exerts a systemic effect on circulating IgG and the plasmatic amino acid profile, in addition to its effect on the colon. Moreover, DSS increases MPO activity in the small intestine and modifies the lymphocyte populations of Peyer's patches, mesenteric lymph nodes, and intraepithelial lymphocytes.

The effects of DSS on the intestinal epithelium occur in a dose-dependent manner. High doses induce severe colitis with diarrhea and colonic mucosal lesions (4,28–30). However, in our study, low doses, such as a 0.5% concentration in drinking water over 5 d, had no severe effects on colonic function; although higher infiltration of neutrophils, greater numbers of goblet cells, hyperemia, and increased water content in feces did occur. Most of these pathological features indicate an epithelial barrier dysfunction in the colonic mucosa. However, unlike protocols using high doses of DSS (6,8,31), our model of intestinal inflammation does not show vacuolation or epithelial destruction, despite increased MPO activity in both the colon and the jejunum, which suggests diffuse neutrophil infiltration. Alteration of mucosal function due to DSS administration also occurred, based on the water content in feces, indicating that DSS-induced mild intestinal inflammation may increase crypt secretion and/or paracellular permeability. High doses of DSS increase the percentage of CD4+ T cells in MLN (32), although this effect was not found using a much lower DSS dose and shorter administration periods, as in our study. It is remarkable, however, that the low DSS concentration induced an increase in the percentage of NK and B lymphocytes in the MLN, in accordance with a significantly higher concentration of IgG in the serum, which supports the view that DSS also has systemic effects.

In the IEL compartment, DSS treatment significantly increased the NK cell population by enhancing the NKR-P1A+CD8– subpopulation. This cell subset has cytotoxic activity and constitutively secretes IFN-{gamma} and IL-4 (33), the latter being a potent regulator of inflammatory processes. Because IL-4 biases inflammation toward a nondestructive, rather than a proinflammatory destructive process (34), increases in this subset in the DSS-treated group could indicate a protective role of these cells at epithelial level when the intestinal barrier is impaired.

Administration of DSS increased the plasma Gln concentration, which was unexpected, given that glutamine is metabolized at high rates by immune cells, particularly in response to antigenic stimuli (35). This observation also differs from studies done in adult rats where no changes in the plasma amino acid profile were observed despite the onset of developing severe colitis (36). Increased plasma Gln concentration induced by DSS may result from lower glutamine incorporation by peripheral tissues, perhaps due to the higher sensitivity of young rats (vs. adult) to the drug.

The administration of glutamine increased the concentration of circulating glutamine by 35% compared with the pair-fed control rats, and similar results were obtained when G-DSS and C-DSS rats were compared. Furthermore, a significantly increased availability of Cit and Arg was detected in all glutamine-supplemented rats. Because Cit and Arg are synthesized from Gln during intestinal catabolism (37), this result may indicate that enterocytes consume glutamine at higher rates when its luminal concentration is increased. The other differences in plasma amino acid concentrations between glutamine-supplemented and pair-fed rats were due to the higher concentrations of nonessential amino acids included in the control diet to meet the nitrogen requirement. The increased plasma concentration of Thr in the Gln supplemented groups can be the result of the diet-induced low plasma Gly concentration affecting the disposal of Thr, as observed by Wilkinson et al. (38) in Arg supplemented pigs. Therefore, although no significant differences were detected in the total amount of amino acids, the glutamine diet enhanced the ratio of essential to nonessential amino acids in rats from the G and G-DSS groups. Other studies have reported that prophylactic dietary glutamine offers protection against intestinal damage, improves nutritional status, and decreases bacterial translocation in experimental models of colitis (15,16,26,27).

Our results show that dietary glutamine supplementation reduced water content in stools from DSS-treated rats. This effect can be due to increased water absorption secondary to increased absorption of luminal osmolytes or to a reduction in paracellular permeability thus reducing water secretion to the luminal compartment. Glutamine is absorbed by Na+-dependent mechanisms (39) which results in osmotic gradients across the epithelium that favor water absorption. However, the amount of dietary amino acids in the control groups was close to that of glutamine in the G groups and they are also absorbed by Na+-dependent mechanisms. Therefore, differences in water absorption between the C and G groups due to differences in luminal Na+ and amino acid absorption rates should not be expected. There are, however, reports indicating that glutamine can protect tight-junction disruption caused by different agents (40); glutamine can also prevent endotoxin-induced increased permeability in piglet ileum without affecting MPO activity (40). These observations are consistent with reported advantages of L-glutamine in oral rehydration therapies, observed in children with acute diarrhea and attributed to a local beneficial effect of glutamine on the mucosa (41) and altogether support the notion that this amino acid has a role in keeping the mucosal integrity.

Rats supplemented with glutamine exhibited significantly higher plasmatic concentrations of Cit and Arg. Because both amino acids are NO donors, and because NO is an inhibitor of leukocyte activation as well as a scavenger of the free radicals produced by neutrophils (42), we hypothesized that the mucosa of the G-DSS group would develop a less aggressive inflammation than the C-DSS group. However, after 5 d of DSS treatment, MPO activity was similar in all DSS-treated rats (jejunum or colon), indicating that the cellular response mediated by neutrophils and macrophages were similar in all groups.

Our observations regarding lymphocyte populations provide new information about the immunological events that take place during DSS administration. The low doses of DSS used in this study resulted in the observation of some mild alterations not yet described in this model of colitis that may prove important for the pathogenesis of the disease. Finally, our study indicates that glutamine supplementation can reduce intestinal luminal water contents during mild inflammation, probably improving intestinal/epithelial barrier function.


   ACKNOWLEDGMENTS
 
The authors thank Ms. Mar Crespí and Dr. Anna Pérez-Bosque for their valuable contributions during the study.


   FOOTNOTES
 
1 This work was supported by project FBG 303352 (Fundació Bosch i Gimpera, Universitat de Barcelona). Back

2 Author disclosures: M. Vicario, C. Amat, M. Rivero, M. Moretó, and C. Pelegrí, no conflicts of interest. Back

3 A color version of Figure 2 is available with the online posting of this paper at jn.nutrition.org. Back

6 Abbreviations used: C, control; DSS, dextrane sulphate sodium; FCS, fetal calf serum; G, glutamine; IBD, Inflammatory bowel disease; IEL, intraepithelial lymphocytes; MLN, mesenteric lymph nodes; MPO, myeloperoxidase; NK, natural killer; PP, Peyer's patches; UC, ulcerative colitis. Back

Manuscript received 7 November 2006. Initial review completed 18 December 2006. Revision accepted 31 May 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 

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