Diarrhea and undernutrition are major health problems in Bangladesh, and both of these conditions are associated with disrupted intestinal mucosal function, as assessed by sugar permeability tests (1–5). Intestinal mucosal biopsies of severely undernourished children show variable degrees of villus atrophy, characterized by shortened, leaf-shaped villi, crypt hyperplasia, and epithelial lymphocyte infiltration (6). Villus damage results in compromised small intestinal barrier function and absorption of intact macromolecules, which may trigger local and systemic immune or inflammatory processes, both of which may further impair intestinal absorption and nutrient use (7).
The lactulose/mannitol (L/M) intestinal permeability test is a noninvasive marker of intestinal absorptive surface area and paracellular permeability (8), which can be used as an indicator of intestinal mucosal function (9,10). The L/M test measures passive absorption and urinary excretion of 2 orally administered nonmetabolizable sugars, 1 of which (mannitol) has a relatively low molecular weight of 182 Da, whereas the other (lactulose) has a higher molecular weight of 342 Da (11,12). Mannitol is assumed to enter the mucosal cell through the hydrophilic portion of the cell membrane, whereas lactulose permeates through the tight junctions and extrusion zones of the intervillous spaces. Consequently, mannitol absorption (as assessed by urinary excretion) can be considered an indicator of the mucosal absorptive area, and lactulose absorption as a measure of the integrity of intestinal mucosal tight junctions (13). Permeability tests using these 2 test sugars assess both aspects of mucosal function, and the use of their urinary recovery ratio provides the additional advantage of controlling for extraneous factors such as gastric retention, liver disease, or partial intraintestinal or urinary bacterial degradation of the sugars, which may otherwise confound the test results (14,15).
Altered intestinal permeability has been documented to occur during and after acute diarrhea (3,15,16) and in children with kwashiorkor and growth faltering (17), iron deficiency (3,18), and zinc deficiency (19). Studies (3) have also found positive associations between altered intestinal permeability and infant age, undernutrition, and nonbreast-feeding. During longitudinal studies, Lunn et al (1) found a significant relation between abnormal intestinal permeability and growth faltering in infants, suggesting either that altered permeability may have contributed to malnutrition or poor nutritional status may have induced changes in intestinal function. However, the causal direction of this relation has remained elusive. We therefore took advantage of a recently conducted 3-month treatment study of severely underweight children to evaluate selected components of the treatment regimen, namely food supplementation and psychosocial stimulation (PS), on changes in the children's intestinal permeability. The study was also designed to compare the L/M in severely underweight and nonmalnourished children.
PATIENTS AND METHODS
The study was conducted at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDRB) Hospital in Dhaka, Bangladesh, from September 2005 to June 2007 in a subset of children who were enrolled in a randomized (open) trial designed to assess different components of an outpatient treatment regimen for severely undernourished children who were previously hospitalized for treatment of diarrhea or other infections. A total of 507 severely underweight children (weight-for-age z score [WAZ] < −3 in relation to the World Health Organization 2006 standard) ages 6 to 24 months were enrolled in the main study, the results of which have been reported separately (20). Children with edema or severe wasting (originally defined as weight-for-length <70% in relation to the National Center for Health Statistics reference data) were treated as inpatients and excluded from the trial, although 35% of the children had moderate wasting (weight-for-length z score [WLZ] <−2) and 47% had severe wasting (WLZ <−3) in relation to the newer WHO standards, which became available only after the trial was initiated.
The original study comprises 5 randomly assigned treatment groups, including a control group that was managed at a hospital-based clinic. The present study only enrolled children who were assigned to 1 of the 4 community-based follow-up groups. The specific treatments assigned to these 4 groups, and the number of children randomly selected for enrollment in the intestinal permeability substudy, were as follows: group-C (control group)—fortnightly follow-up at a community nutrition clinic, including growth monitoring, health education, and micronutrient supplementation (multivitamin drops containing vitamin A palmitate 5000 IU, vitamin D 1000 IU, thiamin 1.6 mg, riboflavin 1 mg, pyridoxine 1 mg, nicotinamide 10 mg, calcium D-pantothenate 5 mg, and ascorbic acid 50 mg/1 mL—1 mL/day; zinc—10 mg/day as zinc sulfate; and iron—3 mg elemental iron/kg body weight/day as ferrous fumarate), n = 23; group-SF—follow-up as per group-C plus supplementary food (SF), n = 26; group-PS—follow-up as per group-C plus PS, n = 23; and group-SF + PS—follow-up as per group-C plus both SF and PS, n = 25.
The SF packets for the SF groups were distributed on the day of recruitment and at each subsequent follow-up visit. Children 6 to 11 months of age were provided with 1 packet per day, and children 12 to 24 months old were given 2 packets per day. Each packet contained 20 g toasted rice powder, 10 g toasted lentil powder, 5 g molasses, and 3 g soybean oil to provide a total of ∼150 kcal per packet, with 11% energy from protein. This food supplement is used in the national nutrition program for community-based management of underweight children. PS consisted of child stimulation and parental counseling conducted by trained health workers (HW). Child stimulation included a half-hour play session conducted by the HW with the children and their mothers during each clinic follow-up visit, using low-cost, culturally appropriate, homemade toys. The mothers were encouraged to continue providing similar stimulation to their children at home. During clinic visits the HW also provided half-hour structured lessons on child development.
For the sake of comparison, we also recruited from the same communities 17 nonseverely underweight children (WAZ and WLZ >−2) who had been free from illnesses for at least 1 week to participate in 1 L/M test of intestinal permeability.
The present study was approved by the Research Review Committee and the Ethical Review Committee of ICDDRB and the institutional review board of the University of California, Davis. Written informed consent was obtained from a parent or guardian of each of the participating children.
The L/M tests of intestinal permeability were completed at the outpatient department of ICDDRB Hospital under the supervision of research staff on the first day the children were enrolled in the study, and the tests were repeated after the children completed 3 months of treatment. For each test, the children received 3 mL/kg body weight of the sugar solutions, containing 400 mg lactulose (Osmolax, Square Pharmaceutical Ltd, Dhaka, Bangladesh) and 100 mg mannitol/3 mL (Sigma, St Louis, MO). Thirty minutes later, the children were allowed to resume breast-feeding and consume their usual meals, except that fructose-containing foods, such as fruit, fruit juice, or any beverages sweetened with fructose corn syrup, were not allowed during the 5-hour test to avoid possible interference with analysis of the test sugars. All of the urine was collected in adhesive, sterile pediatric urine collection bags for 5 hours postdosing. Each time the child urinated, the urine volume was measured and recorded, and the specimen was added to a collection bottle containing 1 drop of 20% (wt/vol) chlorhexidine gluconate to prevent bacterial growth. A 3-mL aliquot of the pooled urine sample was stored at −20 °C until laboratory analysis. Urinary lactulose and mannitol concentrations (milligrams per deciliter) were measured along with known standards in the Nutritional Biochemistry Laboratory of ICDDRB using automated enzymatic methods, as described previously (21), and a Hitachi-902 autoanalyzer (Roche, Mannheim, Germany). The coefficients of variation for the lactulose standards (12.5, 25.0, and 50.0 mg/dL) were 5.9%, 4.8%, and 4.0%, respectively, and for the mannitol standards (25 and 50 mg/dL) 2.3% and 1.8%, respectively. We also spiked samples with the standards to check recovery. Recovery for lactulose was 97.5% to 101.8%, and recovery for mannitol was 95.7% to 100%.
At baseline and at the end of the third month of study a research assistant measured the children's nude weight using a digital scale with 10-g precision (Seca, model-345, Hamburg, Germany); recumbent length to the nearest millimeter using a locally constructed length board; and mid-upper arm circumference (MUAC) to the nearest millimeter using a nonstretchable insertion tape. The research assistant also measured the mothers' weights and heights using standard procedures (22). All of the measurements were taken twice, and the average was recorded; if the measurements varied by >100 g for weight or 5 mm for length/height, or 2 mm for MUAC, a third measurement was taken, and the average of the nearest 2 measures was recorded.
Sample Size Calculation
The planned sample size of 20 children per group was estimated to be sufficient to detect a groupwise difference of 0.09 in the change in L/M, which is half of the difference between the L/M ratio of previously studied nonseverely malnourished Bangladeshi children (19) and severely underweight Brazilian children (23), using a significance level of 0.05 and power of 0.80. We therefore randomly selected 25% (∼25) children from each of the intervention groups to allow for up to 20% attrition after the 3-month period of study. We also completed an L/M study in 17 nonmalnourished children who resided in the same community to compare their baseline L/M data with those of the severely underweight children.
Data were entered and analyzed using SPSS for Windows (version 10.2; SPSS Inc, Chicago, IL). Baseline characteristics were compared among treatment groups and between the malnourished and nonmalnourished children. Outcome variables were compared both within and between treatment groups to assess possible differences in relation to treatment. Categorical variables were compared by χ2 test, or Fisher exact test when the expected number in any cell was ≤5. Continuous variables were compared by analysis of variance followed by Tukey multiple pairwise comparisons. Variables not normally distributed were transformed or compared by Kruskal-Wallis test followed by Mann-Whitney U test. After treatment, 2-factor analysis of covariance was done using SF and PS as main effects, along with their interaction, and controlling for the respective baseline values for urinary clearance of lactulose and mannitol or the L/M ratio and anthropometrics, to examine the effect of treatment group on the respective final values. Results from all of the randomly selected children were included in the analyses on an intention-to-treat basis, and a P value <0.05 was considered statistically significant. The baseline data for the 77 severely underweight children were compared with those of the 17 nonmalnourished children by independent t test or Mann-Whitney U test (as necessary) for continuous variables, and by χ2 test or Fisher exact test (as appropriate) for categorical variables.
A total of 97 severely underweight children completed the baseline intestinal permeability study. Twenty of these children dropped out of the study during the course of the 3-month observation period (group-C = 6, group-SF = 3, group-PS = 6, and group-SF + PS = 5) (Fig. 1). The children who defaulted did not differ with regard to initial age, anthropometrics, or baseline urinary lactulose and mannitol recovery compared with children who completed the study (data not shown), although the L/M ratio was slightly higher among the children who subsequently defaulted (median [interquartile (IQ) range]: 0.19 [0.14–0.35] vs 0.15 [0.09–0.26], P = 0.04). Paired L/M test results (pre- and postintervention) were available for 77 children, 43% of whom were girls. These children's ages, WAZ, length-for-age z score (LAZ), WLZ, and MUAC are shown in Table 1. The children were severely underweight and there were no significant differences in baseline age or anthropometry by treatment group.
The children's weight gain, linear growth, and change in anthropometric indices following treatment are displayed by study group in Table 2. On an average, children in all of the groups gained weight and length during the course of the treatment period. Children who received SF tended to gain more weight, as was the case in the larger study (20), but these differences were not statistically significant for the smaller subset of children included in the intestinal permeability substudy (P = 0.37). There were no groupwise differences in linear growth. At the end of the study, 69% of children were still severely underweight, 8% were severely wasted, and 35% were moderately wasted.
The baseline and final values for urinary recovery of lactulose, mannitol, and their ratios are shown in Table 3 for the severely underweight children, along with the comparison data for the nonmalnourished children. At baseline, the urinary mannitol recovery was significantly less and the L/M ratio was significantly greater among the malnourished children than in the better-nourished comparison children (P = 0.014 and P < 0.001, respectively), but there was no significant difference in mean urinary lactulose recovery. Following 3 months of treatment, the urinary mannitol recovery increased and the urinary L/M ratio decreased significantly among the malnourished children (P < 0.001), but there were no significant differences in these changes by treatment group. Similarly, in the factorial model there were no significant effects of food supplementation or PS on changes in urinary clearance of lactulose, mannitol, or the L/M recovery controlling for the respective baseline values analysis of covariance.
On average, 9.3% of the children had an episode of diarrhea reported during the 14 days before any of the scheduled biweekly follow-up visits, although there were no differences in these intercurrent episodes of diarrhea by treatment group. Ten of the children had an episode of diarrhea reported within 14 days of their final intestinal permeability test, and they had less improvement in their urinary mannitol and urinary L/M recovery ratio than children who did not have a recent episode of diarrhea, although these differences were not statistically significant (mean changes from baseline 0.57 vs 1.52, P = 0.35 and 0.06 vs 0.09, P = 0.75, respectively).
There was a significant positive correlation between the children's change in body weight during the course of treatment and their degree of improvement in the urinary L/M recovery ratio (Fig. 2). A urinary L/M recovery ratio <0.07 is generally accepted as an appropriate cutoff to indicate normal intestinal permeability (3,24). Of the 77 children with complete L/M study results at both time points, 84% had abnormal permeability test results at baseline and 60% still had abnormal results after 3 months of treatment. Children who had normal intestinal permeability results following treatment had significantly greater weight gain (median [IQ range]: 0.92 [0.67–1.32] vs 0.75 [0.47–0.96] kg; P = 0.038) and changes in WLZ (median [IQ range]: 0.69 [0.49–1.97] vs 0.62 [0.22–0.91]); P = 0.047] compared with those whose final L/M ratio was ≥0.07 (Table 4).
Using regression analysis, we examined factors that were associated with a change in the respective urinary sugar recoveries and L/M recovery ratio, including age, baseline anthropometrics and change in anthropometrics, breast-feeding status, and recent episodes of diarrhea and treatment group. The change in WLZ was positively associated with the improvement (ie, reduction) of L/M ratio (β = 0.386, P = 0.01), and the children's age was negatively associated with improvement (ie, increase) in mannitol recovery (β = −0.453, P = 0.019).
The results of the present study confirm previously published observations indicating that severely malnourished children have impaired intestinal mucosal function compared with better-nourished children from the same environment (3,17) and that malnourished children's intestinal function improves during the course of nutritional rehabilitation. The present study results also suggest that recent diarrhea contributes to abnormal intestinal permeability, as has been reported previously (3,15,16). Finally, the present study provides new information by examining whether the recovery of intestinal function is related to differences in selected components of the treatment regimen, such as SF and PS. Contrary to our original hypothesis, the results indicate that there were no specific effects of SF or PS on intestinal mucosal recovery.
Possible reasons for the absence of any observed effects of treatment are that the dietary regimen was inadequate with regard to quantity or nutrient content; alterations in intestinal permeability do not respond to nutritional interventions in general; intestinal mucosal recovery is related only to micronutrient supplementation, which was provided to the children in all 4 treatment groups; or intercurrent illnesses further disrupted the children's intestinal function. The food supplements that were given to children in groups C-SF and C-SF + PS contained only 150 to 300 kcal/day, depending on the age group of the children. Although this amount of food was sufficient to promote slightly greater weight gain among supplemented children in the larger study population (20), this small difference in weight gain may have been inadequate to stimulate major differences in intestinal mucosal recovery. Previous studies have shown that mucosal recovery may be related to specific micronutrients, including zinc and iron (3,18,19), and all children in the present study received multiple micronutrient supplements, regardless of study group. Thus, the children's response to the multiple micronutrient supplements may have overwhelmed our ability to detect a specific response to the SFs. Finally, because the children were treated as ambulatory patients in their own communities, they were continuously exposed to environmental contamination and recurrent enteric infections. Indeed, children reportedly had diarrhea in the previous 1 to 2 weeks during ∼9% of their follow-up visits, and those with a recent episode of diarrhea at the time of their last scheduled visit were less likely to demonstrate improved intestinal permeability. Thus, recurrent infections may have prevented full intestinal recovery despite the more intensive forms of treatment.
Mannitol uptake by the intestine was affected more than lactulose uptake in the malnourished children, suggesting that the major intestinal lesion was loss of mucosal surface area. This is consistent with the results of microscopic examinations, which indicate that malnourished children have shortened villi (6,25,26). Although mannitol uptake increased in all of the groups during treatment, this was not affected by the inclusion of SF in the treatment regimen.
It is interesting that children who demonstrated greater improvement in intestinal permeability results also gained more weight, which is similar to the results of longitudinal, community-based studies in the Gambia (1,27). However, because intestinal recovery was unrelated to treatment group in the present study, it is not possible to determine whether children gained more weight because of their improved intestinal function or vice versa.
A limitation of the L/M test is that it is an indirect measure of intestinal mucosal permeability. Moreover, other simple sugars, such as fructose, can interfere with the test results (27). However, we did not allow the children to consume any substances containing fructose, such as fruits, fruit juice, soft drinks, or any drink sweetened with fructose corn syrup, during the 5-hour urine collection period. Bacterial contamination of the urine can result in abnormal L/M values, but we collected the urine in sterile collection bags and treated the samples with chlorhexidine gluconate as soon as the specimens were obtained, and stored the samples at −20 °C until analysis. Thus, it is unlikely that bacterial contamination affected the present results. As suggested by others (14), we focused mainly on the L/M ratio because the ratio overcomes possible confounding factors, such as the completeness of ingestion of the test sugars, rates of gastric emptying and intestinal transit, possible dilution by intestinal secretions, differences in renal clearance, and completeness of urine collection, which may influence the urinary recovery rate of a single probe (lactulose or mannitol).
In summary, we found that underweight Bangladeshi children were more likely to have abnormal intestinal mucosal function, and possibly decreased mucosal surface area, than their better-nourished counterparts from the same communities. Moreover, children with altered mucosal permeability gained less weight during the period of nutritional rehabilitation. However, it is not certain whether altered intestinal permeability was responsible for impaired nutrient absorption and poor weight gain or vice versa, so additional studies are required to elucidate these relations.
The authors gratefully acknowledge the study donors for their support and commitment to the Centre's research efforts. The authors sincerely appreciate the statistical assistance of Janet M Peerson, statistician of the Program in International and Community Nutrition, and the advice of Dr Rahman Azari of the Department of Statistics and Dr Lucia L. Kaiser of the Department of Nutrition of the University of California, Davis.
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Keywords:Copyright 2010 by ESPGHAN and NASPGHAN
food supplementation; intestinal permeability; lactulose/mannitol test; nutritional rehabilitation; psychosocial stimulation; severe malnutrition