The relationship between malnutrition and child mortality has been emphasized in many studies (1-7), particularly its role in susceptibility to infection (8-12). There is considerable evidence that malnutrition also affects cognitive development, reproduction, physical work capacity and risks for several chronic diseases of adulthood (13-15).
Most children admitted for hospital treatment of severe malnutrition have a history of acute or chronic diarrhea. Both malnutrition and persistent diarrhea have been associated with disrupted intestinal barrier function, which may contribute to the nutrient deficits of these children (16-19). Intestinal mucosal biopsies of severely malnourished children show variable degrees of villus atrophy, crypt hyperplasia and epithelial lymphocyte infiltration (20). Villus damage may cause a loss of mucosal enzymes required for digestion and absorption and may result in compromise of the small intestinal barrier function with the potential side effect of absorption of intact antigenic macromolecules, which may trigger local and systemic immune or inflammatory processes (21). The lactulose/mannitol intestinal (L/M) permeability test is a measure of paracellular permeability and absorptive surface (22). Some studies have shown that supplemental glutamine dipeptide positively influences nitrogen excretion, immune status, gut integrity, intestinal mucosal functions, morbidity and outcome in subjects with diarrhea (23-27). Furthermore, glutamine or alanyl-glutamine stimulate crypt cell proliferation in normal human ileum and colon (24). In patients with inflammatory bowel disease or neoplasic disease, glycylglutamine supplementation has been found to maintain intestinal permeability and villus height (26).
We studied the effects of a modified World Health Organization (WHO) formula supplemented with glutamine on intestinal barrier dysfunction and weight gain in malnourished hospitalized children comparing it to glycine supplemented or unsupplemented formula.
The study was performed at Hospital Infantil Albert Sabin, a 330-bed tertiary medical facility in Fortaleza (population, 2.6 million), in the state of Ceará, Brazil. We studied moderately and severely malnourished hospitalized children between 2 and 60 months of age with or without diarrhea. The weight-for-age z-scores were less than −2 (more than 2 standard deviations below the median for the United States National Center for Health Statistics) (28). Informed consent was obtained from parents for each child's participation. From December 1996 to April 1999, 27 enrolled children received standard formula. From June 1, 2001 to June 30, 2002, 53 patients were randomly assigned to receive in double-blind fashion standard formula supplemented either with glutamine (mw 146 Da; 16.2 g/day) or glycine (mw 75.07 Da; 8.3 g/day) for 10 days. Randomization was achieved by means of computer generated random numbers. Odd or even numbers from this list were used to assign children to receive glutamine or glycine. Randomization was concealed so that the study investigators, physicians, nurses and medical caregiver staff were not aware of the supplement assignments. The individual preparing the glutamine or glycine supplements was not involved in data collection. The daily doses of glutamine or glycine were chosen to equal the molar equivalent of glucose (111 mM) in 1 liter of oral rehydration solution. Glycine was used as a comparison for glutamine because this amino acid is not considered a major metabolic fuel for enterocytes. Isomolar glutamine or glycine was given daily in three divided doses mixed with formula. The primary outcome was intestinal barrier function, measured by the lactulose/mannitol test, and the secondary outcome was weight gain. The protocol was approved by the Clinical Research Ethics Committees of the Federal University of Ceara and the University of Virginia.
After signed parental consent was obtained, we completed a standardized questionnaire recording patient identification number, treatment number, demographic and clinical information, weight and height measurements, results of permeability tests, blood biochemistry test results ordered by hospital physicians and results of stool exams for ova and parasites. The first treatment day started between 7-9 AM the morning after admission and test formula was given for 10 days. The inclusion criteria were as follows: a) age 2-60 months with a history of persistent diarrhea defined as three or more unformed stools/day for ≥14 days or a weight-for-age z-score less than −2; b) agreement to a 10-day hospitalization at Hospital Infantil Albert Sabin; and c) consent of parent or guardian. Children were excluded if they were: a) exclusively breast-fed, preventing adequate assessment of intake; b) intolerant to cow milk; c) participants in any other study within the previous 2 years; d) affected by severe infection or other illness at the time of screening including, but not limited to, shock, meningitis, tuberculosis and varicella; or e) affected by other systemic disease at the time of screening including, but not limited to, tumor and endocrine disorders. Patients discontinued participation for the following reasons: a) patient or guardian requested discontinuation from study; b) patient developed complicating illnesses needing treatment outside of the research unit, such as meningitis, tuberculosis or varicella; c) any unanticipated safety concerns; d) occurrence of serious adverse effects such as vomiting, abdominal distension, watery diarrhea associated with feeding regimen or other unexpected or severe adverse reactions, including death; and e) recurrence of dehydration after initial rehydration.
Standardized Nutritional Support
One group of children received a modified WHO formula standard in our hospital for therapy of malnutrition. The other two groups received formula supplemented with either glutamine or glycine. Formula feeding was started after admission as soon as the child was rehydrated. Supplemented or non-supplemented formula was given every 3 hours. The characteristics of the formulae used are shown in Table 1. We used the following re-feeding program. Children were given 130 ml/kg/day of MF-75 containing 75 kcal per 100 ml until the appetite returned (usually within 2-7 days). If children failed to take at least 80 kcal/kg/day, a nasogastric tube was used. When appetite returned, children were given 130 ml/kg/day of MF-100 containing 100 kcal/100 ml for 2 days, after which feeding volume was increased by 10 ml per feeding to a maximum of 200 ml/kg/day. According to routine practice at Hospital Infantil Albert Sabin, children received a single dose of vitamin A (<6 months, 50,000 IU; 6-12 months, 100,000 IU; and >12 months, 200,000 IU) on the first day. Folic acid, 5 mg on the first day and 1 mg/day thereafter, was also given.
Children without signs or symptoms of infection other than diarrhea were given sulfamethoxazole 25 mg/kg and trimethoprim 5 mg/kg orally in two divided doses daily every 12 hours for 5 days.
Cups for collecting stool specimens from study participants were distributed to the auxiliary nurses. The team was instructed to collect the first stool passed the day after admission. All stool specimens were transported on ice and processed within 4 hours of collection. Bacterial cultures were not performed because all children had received antibiotics as described earlier. All specimens were examined initially by microscopy for ova and parasites and for leukocytes using iodine-stained and methylene blue-stained wet-mount preparations, respectively (30). Reducing substances (modified Clinitest, Bayer Corporation, Elkhart, IN) (31), qualitative fecal fat (32) and fecal occult blood (33) were examined in all stool specimens. Fecal lactoferrin was measured using a Leuko-test kit (Techlab, Blacksburg, Virginia). A fecal parasite concentrator (Evergreen Scientific, Los Angeles, CA) was also used to facilitate parasite examination. A modified acid fast stain was used for detection of Cryptosporidium (34).
Blood analyses on the day of admission were performed in the Central Hospital Infantil Albert Sabin Clinical Laboratory and included hemoglobin concentration, hematocrit, serum concentration of albumin, globulins, glucose, urea and creatinine.
Intestinal Permeability Test
After initial hydration, a lactulose/mannitol test was performed by the method of Barboza et al. (35). After a 3-hour fast, children ingested an aqueous solution (2 ml/kg; maximum volume 20 ml) of lactulose (200 mg/ml) and mannitol (50 mg/ml) and were not fed for 1 hour thereafter. Urine was collected for 5 hours in a flask containing 1 ml of chlorhexidine (40 mg/ml; Sigma Chemical, St. Louis, MO). Total 5-hour urine volume was recorded and a 5-ml aliquot was stored at −20°C for sugar determination by high-performance liquid chromatography with pulsed amperometric detection.
A urine sample (50 μl) was mixed with 50 μl of a solution containing melibiose (3.6 mM) diluted in 2.9 ml of twice-distilled and deionized water. After centrifugation 50 μl was used for sugar determination by high-performance liquid chromatography with pulsed amperometric detection. The BioLC carbohydrate analyzer high-performance liquid chromatography system was composed of a Module GPM-2 gradient pump, a Module EDM-II eluent degassing device and a PAD-II pulsed amperometric detector with a gold working electrode (Dionex, Sunnyvale, CA). A CarboPac MA-1 anion-exchange column (250 × 4.0 mm inner diameter, pellicular resin) with an associated guard column was also from Dionex. The sugars were eluted with an isocratic eluent of 480 mM NaOH at a flow-rate of 0.4 ml/min, with the column kept at room temperature. Sugars were determined with a pulsed amperometric detector as previously described (35). The samples were injected automatically (AS40Automated Sampler; Dionex) and the analyses were quantities using a BioAutoIon 450 Data System (Dionex).
The ratio of urinary excretion of lactulose (marker for mucosal damage) divided by urinary excretion of mannitol (marker for mucosal absorptive area) was used as the primary parameter to measure intestinal barrier function.
Children were weighed daily, without clothes before the first meal, to the nearest 0.01 kg using a calibrated digital scale (Filizola Co., S. Paulo, Brazil). Length was measured supine to the nearest 0.1 cm using a standard anthropometry board at admission and after 10 days on the study protocol. Weight-for-age, height-for-age and weight-for-height Z scores were calculated using EpiNut (World Health Organization, Geneva; Epi Info version 6.0, Centers for Disease Control and Prevention, Atlanta) (28). Kwashiorkor (protein-calorie malnutrition) was diagnosed if there was a characteristic combination of inadequate growth, loss of muscle tissue, increased susceptibility to infection, edema, dermatitis and sparse hair, with or without dyspigmentation (36). Marasmus (inanition) was diagnosed if there was failure to gain weight with muscle atrophy, hypotonia, loss of skin turgor and loss of subcutaneous fat (wrinkled and loose skin with a wizened face) (36). The secondary nutritional parameter used in the clinical protocol was weight change (in grams) after 10 days of treatment.
Sample Size Calculation
Lactulose/mannitol ratio was selected as the primary outcome variable because it is an objective measurement reflecting overall intestinal barrier function and surface area (19,20,35,37). Based on data from previous studies at Hospital Infantil Albert Sabin (38) we expected that children treated with glutamine-supplemented formula would have 30% reduction in lactulose/mannitol ratio compared to those taking nonsupplemented formula. Using a power of 80% and a two-sided significance level of 5%, a sample size of 23 for each group was considered adequate to detect a difference in the lactulose/mannitol ratio between groups. We assumed a possible loss to follow-up of 10% of subjects and thus estimated 26 children in each treatment group.
The data were doubly entered and validated by cross-checking these two databases using Excel software (version 7.0; Microsoft Corporation, Redmond, WA). Data analyses were done using Statistical Package for Social Sciences software version 10.0 (SPSS, Chicago, IL). All variables were tested for homogeneity before applying statistical tests. We used Student's t-test to compare group differences, and categorical variables were tested by χ2 or Fisher's exact test. The lactulose/mannitol ratio and excretions of lactulose and mannitol were log-transformed to normalize these parameters before applying Student's t-test. Linear regression analyses were also done for selected outcomes. All values were two-tailed and P values <0.05 were considered statistically significant.
Twenty-seven children were enrolled in the initial study using standard unsupplemented formula. Sixty children were assessed for eligibility for the supplemented formula trial and 53 (88%) were enrolled. Seven children were not enrolled for the following reasons: two had weight-for-age z-scores greater than −2 and did not meet criteria for at least moderate malnutrition, one was younger than 2 months of age, one had cow milk intolerance, one had low calorie intake, one had hypothyroidism and one had an abdominal tumor. Seventy-seven of 80 (96%) children had weight measurements before and after study. Three children in the nonsupplemented group had incomplete data. Three other children in the nonsupplemented group were considered dropouts (one discontinued at the mother's request, one became septic and one was transferred to another ward for treatment of anemia). One child in the glutamine-supplemented group was diagnosed with varicella on day 10 of the study. Because this child was transferred to another ward after the 10th day, he was not considered a dropout. Sixty-five children had lactulose/mannitol ratios both before and after 10 days of treatment. Eight children in the nonsupplemented group had incomplete data (five did not have the second lactulose/mannitol ratio and three were dropouts). One child in this group had a very high lactulose/mannitol ratio for unknown reasons and this ratio was excluded from final data analysis. Seven children in the supplemented groups did not have both lactulose/mannitol ratios done. Five (three in the glutamine group and two in the glycine group) had samples destroyed in transit to the Clinical Research Unit laboratory, one had a very low mannitol level (glutamine group) and it was presumed that the test solution had been improperly compounded and one (glutamine group) was not tested because he developed varicella on study day 10. Six children had clinical kwashiorkor or marasmus (one in the nonsupplemented group, two in the glutamine group and three in the glycine group). Three of the 80 children (3.8%) had celiac disease (one in the glutamine and two in the glycine group). One child in the nonsupplemented group had cystic fibrosis.
The clinical characteristics of the study children are shown in Table 2. At the start, was no significant difference among groups in age, sex, lactulose/mannitol ratio, weight-for-age, height-for-age and weight-for-height z-scores. All groups had mean weight-for-age z-scores of less than −3, characteristic of moderate to severe malnutrition. In the supplemented formula groups, three of 53 children (9%) had edema at admission (two in the glutamine group and one in the glycine group). A total of 57 (71%) of 80 had three or more liquid stools in the 24 hours before enrollment, 19 of 27 (70%) in the nonsupplemented group, 19 of 26 (73%) in the glutamine group and 19 of 27 (70%) in the glycine group. Most of the clinical histories of diarrhea suggested that the diarrhea was either recurrent or chronic (>30 days duration).
(Table 3) summarizes the number of patients taking MF-75 or MF-100 supplemented diets during the 10 days of treatment in the formula supplemented groups. Twenty-six children received glutamine and 27 received glycine. In the first 2 days of the protocol all children used MF-75. On days 3-6 both MF-75 and MF-100 were used and their proportion was not significantly different between the glutamine and glycine groups. From days 7-10 all children received MF-100.
Caloric intake significant increased during the 10-day period in the glutamine group (r2 = 0.972; P < 0.001) and the glycine group (r2 = 0.965; P < 0.001). Caloric intake was not significantly different in the supplemented groups on any day of the study. On day 10 the mean caloric intake in the supplemented groups were similar (187 ± 56 and 191 ± 67 kcal/kg/day, respectively).
Figure 1 and Table 4 summarize the lactulose/mannitol ratio and the percent urinary excretion of lactulose and mannitol for all groups. In the glutamine supplemented group, the lactulose/mannitol decreased significantly between day 1 and day 10 (0.3129 ± 0.1025 versus 0.1044 ± 0.0175; P = 0.01; n = 21; paired Student's t-test after log-transformation to normalized these parameters), indicating a decrease in permeability. The lactulose/mannitol ratio did not change significantly in the nonsupplemented formula group (0.3842 ± 0.0629 versus 0.3610 ± 0.0613; P = not significant; n = 18; paired Student's t-test after log-transformation to normalized these parameters) or the group supplemented with glycine (0.3703 ± 0.1041 versus 0.1631 ± 0.0358; P = not significant; n = 25). The post treatment lactulose/mannitol ratio was significantly lower in both supplemented groups (Glutamine group P < 0.001; Glycine group P = 0.023; unpaired Student's t-test after log-transformation to normalized these parameters) compared to the nonsupplemented group. The percent urinary excretion of lactulose and mannitol were not significantly different when compared before and after 10 days treatment in any group.
All groups gained weight during nutritional therapy for 10 days. Mean total weight gain in the 24 unsupplemented children with complete weight data was 142 g (initial weight 5131 ± 449 g; end weight 5273 ± 472 g). The mean total weight gain of the 26 glutamine supplemented patients was 213 g (initial weight 6668 ± 535 g; end weight 6881 ± 535 g). The mean weight gain of the 27 glycine supplemented patients was 252 g (initial weight 5673 ± 512 g; end weight 5925 ± 529 g). There was no statistical difference among these mean weight gains although there was a trend toward higher mean weight gains in both supplemented groups. These results did not change when we excluded from the data analyses the children with edema, kwashiorkor or marasmus. These results were also similar when we included only children taking MF-100. We did not observe a significant change in weight-for-age z-scores after treatment in any group.
Stool samples from 60 children (19 in the glutamine group and 17 in the glycine group) were collected on study day 1 for laboratory examination. Twenty-seven of 60 (45%) had liquid stools (13 nonsupplemented, five glutamine and nine glycine) had liquid stools. Occult blood testing was positive in three of 24 children in the nonsupplemented group and was negative in all other patients. Reducing substances were found in two samples (one nonsupplemented and one glutamine). White blood cells were seen in seven samples (six nonsupplemented and one glutamine). Lactoferrin was positive in 32 stool samples (12 nonsupplemented, 12 glutamine and eight glycine). White blood cells were found significantly more often in stools of nonsupplemented children (25%; P < 0.05) than in those given formula supplemented with glycine. Parasites were found in six children. Four nonsupplemented children had T. trichiuris (n = 1) and Cryptosporidium spp. (n = 3). One child given glycine supplement had Entamoeba coli and Ascaris lumbricoides and one child given glutamine had Giardia lamblia. Children with T. trichuris, A. lumbricoides or Giardia were treated with mebendazole or metronidazole, respectively. The parasitologic exam showed no significant differences between the groups at admission.
The mean duration (±SD) of diarrhea not significantly different between the glutamine (n = 19) and glycine (n = 18) groups (9 ± 0.07 days and 8 ± 3.03 days, respectively). Mean hemoglobin and hematocrit were below normal in glutamine group and normal in the glycine group. Mean total serum protein and albumin were below the normal range in both groups. Mean serum globulin concentration was normal in both groups. Serum urea was above the normal range and creatinine was normal in both groups. There were no significant differences between glutamine and glycine groups for any laboratory blood tests. One glutamine supplemented patient was human immunodeficiency virus positive by ELISA test. Nine of 26 (35%) in the glutamine group had abnormal chest radiographs, as did four of 27 (15%) in glycine group (P = not significant). Two patients had pneumonia on admission, one in the glutamine group and one in glycine group.
Although glutamine is considered a nonessential amino acid, its synthesis may not keep up with requirements during times of deprivation or stress, thus making it conditionally essential (39). In children with persistent diarrhea and malnutrition there is a theoretical rationale for supplementation with glutamine or its derivatives, although its clinical utility and biologic significance in children with diarrhea has not been proved. Malnourished children with diarrhea have decreased plasma glutamine concentration, and they often have complicating infections that increase the utilization of glutamine (25,34,38).
Several studies of malnutrition (16,19) and of acute and persistent diarrhea (17,18) have demonstrated a negative impact of these conditions on intestinal mucosal absorptive and barrier function. Ford et al. (40) studied 39 children with diarrhea and 28 control children undergoing duodenal biopsy and found that abnormal intestinal permeability was associated with diarrhea and with mucosal damage. Ford et al.'s report strongly suggested that the dual sugar permeability testing was a reliable and useful index of mucosal integrity and a measure of intestinal mucosal absorptive and barrier function. Lunn et al. (16) studied chronic diarrhea and malnutrition in Gambian children and found evidence of impaired intestinal mucosal absorption and barrier function in these children. Several studies have confirmed these findings and have documented the ability of the dual sugar permeability test to assess intestinal mucosal absorptive and barrier function (19,35,37,41-43). The permeability test is noninvasive, making it a valuable tool for clinical studies evaluating the impact of various therapies in the management of persistent diarrhea and malnutrition.
This study evaluates the effect of nonsupplemented formula and formula supplemented with glutamine or glycine on intestinal barrier function and weight gain in children with severe malnutrition. The children studied were similar in age, gender, initial intestinal permeability studies and nutritional status. Our study is in agreement with other reports (26,45) in which glutamine prevented diminution of intestinal mucosal barrier function in patients on total parenteral nutrition. Yalçin et al. (44) recently conducted a placebo-controlled double-blind, randomized trial of children 6 to 24 months old with acute diarrhea and found a significant decrease in duration of diarrhea in those supplemented with glutamine. This study suggested that the beneficial impact of glutamine was through effects on gastrointestinal mucosal function rather than effects on host immune responses. Ribeiro et al. (46) studied an oral rehydration solution containing 90 mM glutamine and compared its efficacy to standard WHO oral rehydration solution in infants 1 month to 1 year of age with acute noncholera diarrhea and dehydration. We chose our supplement based on the dose of glutamine, the amount of solution intake and safety information from this study. We chose an amount of glutamine (or glycine) equivalent to the molar amount of glucose (111 mM) in 1 liter of standard WHO oral rehydration solution. The concentration of glutamine or glycine in our modified supplemented formula was isosmolar, thus avoiding any effects of osmolality on the permeability test or on diarrheal symptoms (37,47). However, the glutamine and glycine consumed per day contained different amounts of nitrogen, 3.1 g and 1.6 g, respectively. The extra amide nitrogen present in glutamine might be physiologically significant in the synthesis of purines, pyrimidines and glucosamine (48).
The optimal duration of glutamine supplementation is uncertain. Our pilot data suggested that treatment with a smaller dose of glutamine added to regular hospital diet for 5 days prevented barrier function damage (38). Hence we decided to test a longer treatment regimen with an increased amount of glutamine. In both studies glutamine was given enterally. The advantage of enteral supplementation is that glutamine is delivered directly to the intestine (49,50). In in vitro studies in which glutamine was applied to Caco-2 cell monolayers, less bacterial translocation occurred when glutamine was applied to the apical versus the basolateral cell surface (51), suggesting that mucosal administration is the most efficacious route for supplementation. Rhoads et al. (52) found that when rat crypt cells (IEC-6) or piglet enterocytes (IPEC-J2) were cultured in glutamine-free medium and then exposed to glutamine for 24 hours, 3H-thymidine incorporation in DNA increased by more than 20-fold. Exposure of intestinal cells to glutamine appears to activate a signaling pathway mediated by mitogen activated protein kinases within minutes; these include both extracellular signal related kinases and the nuclear kinase c-Jun (53). Although the mechanisms of glutamine effects in clinical studies are poorly understood, we postulate that glutamine may promote enterocyte proliferation.
The results of the current study are consistent with previous work (38,46,54) suggesting that enteral glutamine is safe and well tolerated in malnourished children with or without persistent diarrhea. In this study there were no dropouts as a result of unexpected safety concerns. Our previous work has not shown any abnormal renal or liver function after glutamine and no serious adverse events (38). There were no deaths in this study.
We added enteral glutamine or glycine supplements to a liquid diet based on the milk formulas F-75 and F-100 and given according to the WHO guidelines for the treatment of malnourished children (29). The study groups did not differ in the proportion of days receiving either MF-75 or MF-100. The mean caloric intakes were not significantly different among the groups throughout the study. Both supplemented and unsupplemented groups had a significant improvement in caloric intake as expected during the study. Results of stool examinations were not significantly different between groups; thus we believe that parasitic infection did not bias our results.
In conclusion, we found that enteral formula supplemented with glutamine was associated with an improvement in intestinal barrier function as measured by the lactose/mannitol ratio. We did not observe that supplementation with glutamine or glycine was associated with better weight gains than observed in patients treated with the standard WHO formula and protocol for malnourished children. Our study did not allow any conclusion regarding the impact of enteral supplements of glycine or glutamine on diarrhea.
Acknowledgments: We would like to acknowledge all children and their guardians for their permission to allow their children to participate in this approved protocol by our Ethical Clinical Research Committee. We also acknowledge all participating physicians, nurses, medical caregivers and laboratory technicians for their important work support on this study protocol.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
Malnutrition; Persistent diarrhea; Intestinal permeability; Glutamine; Glycine