Breast milk is optimal nutrition for the infant and also provides unique protection during the early postnatal period when both the immunologic system and gut barrier functions are immature (1,2). Data are accumulating to indicate that infants may be sensitized to dietary antigens even during exclusive breast-feeding (3). This may be because of prenatal exposure (4), antigens present in breast milk (5,6) or the home environment (7), or deficiency of immunologically active components in breast milk (8,9).
Atopic eczema often is the first manifestation of sensitization. Atopic symptoms during breast-feeding raise the question of whether or not to continue breast-feeding in sensitized infants. We have previously demonstrated that symptoms of atopic eczema diminish and the nutritional state affected by allergic inflammation improves after weaning of breast-fed infants with atopy to a tolerated substitute formula (3). However, it is currently not known whether the shift from breast milk to a hypoallergenic formula is beneficial to the gut barrier of an infant with atopy.
The purpose of this study was to evaluate gut barrier functions in infants with atopy during breast-feeding and after weaning to a tolerated hypoallergenic formula. Intestinal permeability was assessed by evaluating the recovery of oral lactulose and mannitol in urine and intestinal protein leak by measuring the concentration of α-1 antitrypsin in feces. Methylhistamine and eosinophil protein X (EPX) excretion into urine were measured as markers of allergic inflammation.
PATIENTS AND METHODS
We studied 56 consecutive breast-fed infants who were admitted to the Department of Paediatrics at Tampere University Hospital, Finland, because of atopic eczema. Inclusion criteria were that atopic eczema fulfilling the Hanifin criteria (10) had begun during exclusive breast-feeding, when they had never ingested anything but breast milk and water. All patients were born after full-term pregnancy. In most cases (89%) there was a positive history of atopic diseases in first-degree family members. Atopic eczema had begun at a mean age of 2.0 months (range, 0.1–6 months). Altogether, 41 mothers eliminated milk products, egg, fish, or cereals from their diet during lactation to alleviate the symptoms of atopic disease in their infants, either by themselves or as a consequence of the advice of a public-health nurse or a general practitioner at the well-baby clinics. However, the maternal elimination diet was not based on formal allergy testing in any infants. Despite the maternal elimination diet, atopic eczema continued and required referral to university hospital. The mean duration of exclusive breast-feeding (not even a single exposure to food other than breast milk) was 3.5 months and that of total breast-feeding before weaning to formula was 5.4 months.
The parents were informed verbally and in writing about the nature and requirements of the study. Their written informed consent was obtained, and the study was approved by the Ethics Committee of Tampere University Hospital. No parents refused to participate in the study.
All patients received the same information, and the follow-up was conducted in uniform manner. The infants were studied at enrollment, at a mean age of 5.0 months (range, 2.0–9.6 months), when they were still being breast fed. By that time, 23 patients had been given small amount of cow's milk-based formula, and 10 had ingested cereals. The follow-up visits were scheduled 2 and 7 months after the cessation of breast-feeding, at the age of 7.4 months (range, 3.9–12.1 months) and 12.6 months (range, 8.2–25.4 months) months, respectively. The researchers did not interfere with the timing of weaning, which was determined by the mothers only.
During each visit the patients were clinically examined and the severity of atopic eczema assessed according to the SCORAD method (11) by the same pediatricians (TA and EI). In evaluating the extent of sensitization, serum total IgE (Phadebas IgE Prist, Pharmacia, Uppsala, Sweden), and specific IgE (radioallergosorbent assay, Pharmacia) and skin prick tests for cow's milk, egg, wheat, oat, barley, and rye were studied. These were selected to represent the most important food allergens in early childhood. The foods suspected of causing allergic symptoms on the basis of clinical history, specific IgE, and skin prick test results were eliminated from the infants' diet and substituted by nutritionally equal foods. After the cessation of breast-feeding, a double-blind placebo-controlled cow's milk challenge was performed in 53 infants, as previously described (12). Three infants were not challenged because of severe symptoms after accidental exposure to a small amount of cow's milk.
No new foods were allowed during the first month after weaning or during the cow's milk challenge period. The weaning diet consisted of extensively hydrolyzed whey formula (Peptidi-Tutteli, Valio Ltd., Helsinki, Finland) or amino-acid derived formula (Neocate, SHS Int., Liverpool, United Kingdom), tolerated by the infant. The changes in the variables used to assess gut barrier were not influenced by the specific formulas or diets during the study. After 5 to 6 months of age, the most commonly used solid foods were potato, rice, cauliflower, berries, and pork, which were gradually introduced to the diet of the infants if no adverse reaction occurred. Moisturizing creams were used several times daily to humidify skin. Topical corticosteroids for 5- to 7-day periods or systemic antihistamines were prescribed when deemed necessary.
Intestinal permeability and urinary concentrations of methylhistamine and EPX were assessed during the symptomatic period when the infants were breast fed and 2 months after weaning to a tolerated hypoallergenic formula. Fecal α-1 antitrypsin was studied before weaning and 2 and 7 months after weaning.
Intestinal permeability test
In all patients this test was performed at the same time of day, starting at 9 am. For ethical reasons the infants were not fasted during the test period but were allowed to drink breast milk or a tolerated hypoallergenic formula (Neocate or Peptidi-Tutteli). Breast milk does not contain lactulose or mannitol (13), nor does the Neocate (information from SHS Int.). Peptidi-Tutteli contains no mannitol but an insignificant amount of lactulose (330 mg/L) (information from Valio Ltd.).
After a baseline urine sample had been obtained, a 100-mL solution containing 4 g (11.7 mmol/L) of lactulose and 0.8 g (4.4 mmol/L) of mannitol (227 mOsm/kg of H2O) was administered orally. Five infants received the solution by nasogastric tube because they refused to drink the solution. All urine passed during the next 5 hours was collected in a plastic urine-collection bag (Urinocol Pédiatrie, B. Braun Biotrol S.A., Paris, France) and measured. An aliquot was immediately frozen and kept at −70°C until analysis.
Lactulose and mannitol concentrations in urine were measured by gas chromatography. Urine samples supplemented with internal standards were first diluted with an equal volume of distilled water and desalted with Amberlite MB 604 resin (BDH, Poole, United Kingdom). An aliquot (100 μL) of the supernatant was evaporated at 60°C under a stream of nitrogen. Thereafter, 75 μL of N,O,bis(trimethylsilyl)-trifluoroacetamide (Pierce, Rockford, IL, U.S.A.) was added and allowed to react for 60 minutes. Derivatized sugars were separated with Rtx-35 column (ID 0.32 mm, length 30 m; Restek Corp., Bellefonte, PA, U.S.A.), and inositol and turanose were used as internal standards to quantitate mannitol and lactulose, respectively. The day-to-day coefficients of variation measured in a pooled urine sample (n = 14) were 2.8% and 3.3% for mannitol and lactulose, respectively. The method was tested to separate lactulose from lactose and saccharose, which may be present in urine samples from unfasted infants.
Analyses of urinary concentration of methylhistamine and eosinophil protein X (EPX)
Methyl-histamine and EPX concentrations in the diluted urine samples were measured by radioimmunoassay using reagents from Pharmacia & Upjohn Diagnostics, Uppsala, Sweden. Results are expressed as micrograms of methyl-histamine or EPX per millimoles of creatinine in the urine sample.
Analysis of fecal α-1 antitrypsin
Approximately 1 g of homogenized feces was lyophilized, and about 50 mg resulting dry material was weighed and transferred to an Eppendorf tube. α-1 antitrypsin was extracted by vigorous mixing with 1 mL of 0.15 M NaCl solution. The resulting suspension was centrifuged at 25,000 g for 10 minutes to remove debris. The supernatant concentration of α-1 antitrypsin was measured using a Behring BN II nephelometer. The results are given as milligrams per gram dry weight of lyophilized feces.
Data are given as means with 95% confidence interval (CI) or medians with interquartile range (IQR). Wilcoxon signed-rank test was used in pairwise comparisons and Spearman rank correlation coefficient to determine the degree and significance of association between urinary EPX concentration and lactulose/mannitol recovery ratio and the SCORAD score.
Relation between weaning and the severity of atopic eczema
During breast-feeding, the mean (95% CI) SCORAD score was 22 (18–27), and the median (IQR) serum total IgE was 9.0 kU/L (range, 0.0–35.5 kU/L). After weaning, the extent, intensity, and subjective symptoms of atopic eczema diminished significantly (Table 1). Table 2 shows the results of specific IgE and skin prick test examinations. Double-blind placebo-controlled cow's milk challenge confirmed cow's milk allergy in 30 of 53 (57%) patients.
Relation between weaning and the concentrations of urinary eosinophil protein X and methylhistamine
During the symptomatic period when the infants were breast fed, the median (IQR) urinary EPX concentration was 101.6 μg/mmol (range, 67.1–171.7 μg/mmol) creatinine. This decreased significantly after weaning (Table 1). The decrease in urinary EPX concentration did not correlate with increasing age. There was a direct correlation between the urinary EPX concentration and the SCORAD score during breast-feeding (rho = 0.32, P = 0.02) and after weaning (rho = 0.30, P = 0.02).
In contrast, the urinary methylhistamine concentration was comparable during breast-feeding and after weaning (Table 1).
Relation between weaning and gut barrier functions
During breast-feeding, the median (IQR) urinary recovery ratio of lactulose and mannitol was 0.029 (range, 0.021–0.042). There was a direct correlation between lactulose/mannitol recovery ratio and the SCORAD score during breast-feeding (rho = 0.37, P = 0.006). This was attributable to a lower urinary recovery of mannitol in patients with higher SCORAD score (rho = −0.40, P = 0.003). After weaning, a significant decrease in the lactulose/mannitol ratio was observed (Table 1). The decrease in the lactulose/mannitol ratio after weaning was associated with a rising trend in urinary excretion of mannitol (P = 0.06), whereas the urinary excretion of lactulose remained unaltered.
In addition, the concentration of fecal α-1 antitrypsin decreased significantly after weaning (Table 1).
We studied a highly selected population of young infants (i.e., those who experienced atopic eczema even before exposure to any food other than breast milk). The predisposition to atopic disease among these infants was high because in almost 90% of cases there was a positive history of atopic disease among first-degree relatives, thus explaining the high proportion of infants with positive skin prick test or specific IgE concentration to food antigens. The results of such examinations have been reported by others (12,14) to be commonly positive in infants with atopic eczema.
The results of the current study demonstrate that in breast-fed infants with atopy, intestinal permeability and the concentrations of fecal α-1 antitrypsin and urinary eosinophil protein X decrease concomitantly with diminishing SCORAD score after cessation of breast-feeding and starting of hypoallergenic formula. Despite decreasing urinary eosinophil protein X concentrations, the concentration of methylhistamine remained unaltered, possibly reflecting the different underlying phenomena (i.e., the release of histamine from mast cells and basophils and release of eosinophil protein X from eosinophils).
Many studies have reported increased macromolecular transport across gut barrier in children with atopy (15,16). The enhanced gastrointestinal permeability is thought to reflect mucosal damage induced by local hypersensitivity reaction to foods (17). Indeed, morphologic abnormalities have been demonstrated in children with atopic eczema (18). We chose to evaluate intestinal permeability by the lactulose/mannitol test, which is nontoxic, noninvasive, and simple to perform. Orally administered mannitol, a small molecule (molecular weight, 182), is thought to cross the epithelium through the cell membrane (19). Lactulose, a larger molecule (molecular weight, 342) is thought to cross the epithelium only through paracellular junctions. In gut inflammation, lactulose transport through the epithelium is increased, whereas mannitol transport remains mainly unaltered or may be decreased if the villous surface is reduced (20,21). The lactulose/mannitol ratio is regarded as a more sensitive marker of altered mucosal integrity than is measure of either of the two sugars alone (21). Thus, lower urinary recovery of mannitol in patients with higher SCORAD score would reflect diminished absorptive surface area in infants with symptoms. The unaltered excretion of lactulose in urine in the patients in the current study suggests that mucosal integrity has remained intact.
We observed a significant decrease in the urinary recovery ratio of lactulose and mannitol after weaning. This could be explained in two ways: first, decreased intestinal permeability during intestinal maturation (i.e., “gut closure”), or second, diminished inflammation and epithelial healing concomitant with ameliorating atopic eczema. The decreasing concentrations of urinary eosinophil protein X and fecal α-1 antitrypsin support the latter concept. The urinary level of eosinophil protein X, which is one of the major granule proteins released by activated eosinophils, has been shown to reflect eosinophilic inflammation (22) and correlate with the severity of atopic eczema in children (23). In addition, a decreasing level of fecal α-1 antitrypsin, a marker of protein loss (24) and inflammation (25) in the intestine, was detected. Our data point to improved gut barrier function after weaning. Although α-1 antitrypsin is present in breast milk, this decrease may not be explained by cessation of breast-feeding alone because previous data show no difference in the concentration of fecal α-1 antitrypsin between formula-fed and breast-fed infants at this age (26).
A lack of healthy infant controls limits the conclusions of our study. However, it is unlikely that the decrease in the urinary recovery ratio of lactulose and mannitol was due to “gut closure” because an age-dependent decrease in intestinal permeability in humans has been demonstrated only during the first few days of life (27,28), whereas the permeability remains stable and low in infants with ages similar to those of our study patients (29,30)
In conclusion, our results indicate that in breast-fed infants with atopy, gut barrier function is improved after cessation of breast-feeding and starting of hypoallergenic formula.
The authors thank Mrs. Annette Tahvanainen, RN, and Mrs. Pirjo Luukkonen for excellent assistance.
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