The incidence of acute pancreatitis (AP) in children has been increasing over the past three decades. Most pediatric cases of AP are mild to moderate. One proposed mechanism of AP involves the reduced integrity of the intestinal mucosa. Intestinal permeability increases early in the course of AP due to splanchnic hypoperfusion and subsequent ischemia-reperfusion injury, which damages the gut mucosa.1,2 This damage may allow bacterial translocation, contributing to systemic inflammation and sepsis.3 Numerous studies have shown increased intestinal permeability in adults with AP,4–10 but its impact in pediatric patients has not yet been evaluated. Although no single noninvasive method assesses intestinal permeability reliably, plasma zonulin is considered a potential marker.11 Studies in adults suggest that early oral feeding may shorten hospital stays and reduce the incidence of infectious complications, morbidity, and mortality.12–17 These findings have influenced pediatric recommendations.18,19

Given that early nutrition may improve intestinal permeability (reflected indirectly by lower plasma zonulin levels) and potentially improve outcomes, we examined the effect of very early enteral nutrition (within 24hours of admission) compared with early nutrition (after the first 24hours) on plasma zonulin concentrations in children with mild-to-moderate AP.

We conducted a prospective randomized study between January 2019 and September 2022 in three university hospitals. It included children aged 1 to 18 years with AP, diagnosed according to standard criteria (meeting at least two of the following: abdominal pain suggestive of pancreatitis; serum amylase and/or lipase levels at least three times the upper limit of normal; characteristic imaging findings) and with no contraindications for enteral nutrition.18 The severity of AP was assessed after 48hours.20 Mild AP was defined as absence of organ failure and local or systemic complications, while moderate AP was defined as transient organ failure, local complications, or exacerbation of comorbidities.20 Patients who developed severe AP were excluded from the analysis. Additional exclusion criteria included severe illness/instability, inflammatory bowel disease, gastrointestinal or respiratory infections, renal or cardiac insufficiency, type 1 diabetes, cystic fibrosis, contraindications to enteral feeding, or lack of informed consent.

Patients were randomized using a computer-generated algorithm in blocks of four into two groups: (A) refeeding within 24hours of admission (very early) and (B) refeeding at least 24hours after admission but within 72hours (early nutrition). Refeeding consisted of a low-fat diet prepared by hospital dieticians and adapted according to the patient’s tolerance. All children received appropriate fluid therapy and pain management as needed.

The primary outcome was intestinal permeability, assessed via plasma zonulin concentrations. Blood samples were collected on the first, second, and third days of hospitalization using EDTA as an anticoagulant. Samples were centrifuged at 2000 g (3575 revolutions per minute) for 10minutes at 4°C and then frozen at −80°C within 2hours of collection. Concurrently, standard biochemical tests were performed in the local laboratories of the participating hospitals, including C-reactive protein (CRP) levels and serum activity of amylase, lipase, and aminotransferases (alanine aminotransferase, asparagine aminotransferase, and gamma-glutamyl transferase). As laboratory methods varied across sites, each CRP result was expressed as a percentage of the upper limit of normal.

We used the IDK Zonulin ELISA kit for the in vitro measurement of zonulin in plasma. Preparation of standards, reagents, and plasma samples, as well as the assay procedure and data acquisition, were carried out according to the manufacturer’s instructions. The method has been shown to be linear from 3.03 to 40.25ng/mL, with nonlinear behavior of less than ±20% within this range for concentrations above the limit of quantitation. The limit of detection was 0.183ng/mL.

The sample size was determined based on previous research showing a difference in length of stay between patients refed with liquid vs nonliquid diets (mean [SD], 6.75 [3.37] vs 4.18 [2.86] days).21 For a statistical power of 80% and a significance level of 5%, we calculated that a minimum of 35 patients were required per group, for a total of 70 patients. Allowing for an estimated 20% rate of non-evaluable cases, the final sample size was set at 84 patients.

The statistical analysis was performed with the software Statistica 13 (StatSoft; Oklahoma, USA). We used the Shapiro-Wilk test to assess the normality of the distribution of quantitative variables. Continuous variables were compared using the nonparametric Mann-Whitney U test, and proportions with the χ2 test, applying the Yates correction when appropriate. We considered p values of less than 0.05 statistically significant. The Spearman correlation coefficient was used to assess the strength and direction of associations between variables.

The study was conducted in accordance with the Declaration of Helsinki for human research and was approved by the ethics committee. We obtained informed consent from the parents or legal guardians of all participants and from patients older than 16 years. The study was registered at ClinicalTrials.gov (NCT03820128). Minor modifications to the registered protocol were made before study initiation, changing measurements from days 1, 3, and 5 to days 1, 2, and 3.

A total of 94 children were recruited for the study. Nineteen patients were excluded because they did not meet the inclusion criteria (Fig. 1). Zonulin concentrations were measured in 61 children: 37 (60.66%) in group A (refeeding within 24hours of admission) and 24 (39.34%) in group B (refeeding at least 24hours after admission). All patients had mild AP.

Figure 1.

Flow chart of study participants.

The groups did not differ significantly in age, weight, height, body mass index, approach to diagnosis of AP, or laboratory results (Tables 1 and 2). Zonulin concentrations were similar in both groups during the first 3 days of hospitalization (Table 3). We found a weak but statistically significant positive correlation between zonulin and gamma-glutamyl transpeptidase concentrations throughout the 3 days (day 1: R=0.291, P= .028; day 2: R=0.309, P= .027; day 3: R=0.331, P= .015).

Zonulin is used as an indirect marker of intestinal permeability. It correlates strongly to the lactulose/mannitol ratio (LMR), a test commonly applied to assess permeability in various gastrointestinal diseases and in malnutrition.22,23 In 1991, Fasano et al. identified zonula occludens toxin (Zot), an enterotoxin produced by Vibrio cholerae that causes a reversible opening of intracellular tight junctions.24,25 In 2000, the same group described zonulin, the eukaryotic counterpart of Zot, whose expression is increased in intestinal tissue during the acute phase of celiac disease.26 Zonulin activates the epidermal growth factor receptor (EGFR), both directly and through transactivation by several G-protein-coupled receptors (GPCRs), including PAR2.27,28 This leads to phosphorylation of target proteins and subsequent polymerization of soluble G-actin into F-actin. The resulting rearrangement of actin filaments causes the displacement of junctional proteins, including zona occludens proteins, ultimately loosening intestinal tight junctions.29

In our prospective study, zonulin levels were comparable between the two groups throughout the first three days of hospitalization.

To date, no studies assessing intestinal permeability in children with AP have been published. In most studies involving adults, permeability was evaluated using LMR, the lactulose-l-rhamnose ratio, or urinary excretion of polyethylene glycol (PEG). These studies consistently found that intestinal permeability was significantly greater in patients with AP compared with healthy controls.4–6 Permeability was also significantly greater in patients with severe AP compared to those with mild disease.6–8,30 Increased intestinal permeability was positively correlated to septic complications, longer intensive care unit stays, prolonged hospitalization, and increased mortality.7,9,10

Zonulin concentrations have also been examined in experimental rat models of AP. Consistent with findings for other permeability markers, zonulin levels were significantly higher in rats with AP than in controls and were more elevated in severe compared to mild AP.31 However, these results were not confirmed in the only clinical study that evaluated zonulin to date, which found no differences in plasma zonulin levels between controls and AP patients or between patients with mild, moderately severe, and severe AP.32 Discrepancies between animal and human studies may reflect differences in the underlying pathophysiological processes, but they may also stem from the small human sample sizes, particularly the group with severe AP (five patients).32

The impact of enteral nutrition on intestinal permeability in AP has gained increasing attention. Enteral feeding helps preserve the gut barrier and reduces bacterial and endotoxin translocation,33 whereas even 48hours of fasting markedly increases apoptosis within the intestinal mucosa.4,34 Li et al. reported that patients with AP who received enteral nutrition after two days of fasting had lower intestinal permeability than those receiving parenteral nutrition.35 Similarly, Shen et al. compared parenteral and enteral nutrition in patients with severe AP and found that intestinal permeability was significantly higher in the parenteral group 1 and 2 weeks after the intervention.36 However, none of these studies evaluated how the timing of enteral nutrition initiation affects intestinal permeability, and the small sample sizes limit the strength of their conclusions.

Our study has several limitations. The number of participants was limited (partly due to the SARS-CoV-2 pandemic); however, it was sufficient for statistical analysis. Because of preanalytical and laboratory errors, zonulin concentrations could not be measured in all patients. The sample size was calculated for a larger project assessing the impact of nutrition on AP, in which the primary endpoint was the length of stay, while zonulin-based assessment of intestinal permeability was a secondary endpoint. The sample only included children with mild to moderate AP, so the impact of very early nutrition on severe AP remains unknown; however, severe AP in children is rare. It should also be noted that plasma zonulin, as an indirect marker of intestinal barrier integrity, may vary between individuals, and factors such as diet or biological sex may affect its concentration.37,38 To obtain a more homogeneous cohort, we excluded patients with conditions that could affect intestinal permeability.

Very early enteral nutrition does not appear to influence intestinal barrier permeability in children with mild pancreatitis compared with early nutrition, based on plasma zonulin levels. These findings contribute to a better understanding of the pathophysiology of AP and the mechanisms by which nutrition may improve outcomes. Indirectly, they provide further evidence supporting the safety of introducing enteral nutrition as early as possible in pediatric patients with mild AP. Further studies are needed to clarify whether plasma zonulin accurately reflects AP activity in humans and to what extent.