Blood, Stool and Urine Tests Could Track Gut Damage in Children With Celiac Disease

Children with celiac disease are usually diagnosed with blood antibody tests and, when needed, a duodenal biopsy, but these tools have well-known blind spots. A mini review published in Frontiers in Pediatrics examines the growing menu of emerging biomarkers that could fill those gaps, with particular attention to pediatric patients. The review, written by researchers at the Republican Specialized Scientific-Practical Medical Center of Pediatrics in Tashkent, Uzbekistan, concludes that two candidates stand out: intestinal fatty acid-binding protein, known as i-FABP, for detecting and tracking intestinal injury, and gluten immunogenic peptides for objectively monitoring whether children are actually sticking to a gluten-free diet. Everything else, the authors stress, remains investigational.

Celiac disease is an immune-mediated enteropathy triggered by gluten in genetically predisposed people who carry the HLA-DQ2 or HLA-DQ8 variants, affecting roughly 1% to 1.5% of the global population, with a rising incidence in children. Current diagnostic algorithms from the European Society for Paediatric Gastroenterology, Hepatology and Nutrition combine clinical assessment with IgA antibodies against transglutaminase 2 and endomysial IgA antibodies, and biopsy can be omitted in selected patients whose anti-TG2 IgA levels exceed ten times the upper limit of normal alongside positive endomysial antibodies. But the approach falters in IgA deficiency, minimal mucosal lesions, potential disease and seronegative cases, and the available serological markers correlate poorly with histological recovery once treatment has begun.

i-FABP is a small cytoplasmic protein expressed in mature villus-tip enterocytes, the absorptive cells most damaged by celiac disease. Because of its low molecular weight of roughly 15 kilodaltons, it leaks into the bloodstream as soon as enterocyte membranes are injured and is cleared by the kidneys, which also allows detection in urine. According to the review, serum i-FABP is consistently elevated in children with newly diagnosed celiac disease, correlates with the severity of villous atrophy graded on the Marsh scale, and declines rapidly after a gluten-free diet begins, often before conventional serology normalizes. Experimental gluten-challenge studies show it rising within days of gluten ingestion, ahead of changes in anti-TG2 antibody titers.

The marker has real limitations. i-FABP is not specific to celiac disease and is also elevated in inflammatory bowel disease, intestinal ischemia and necrotizing enterocolitis, and diagnostic thresholds vary across assays, so it cannot stand alone. Urinary measurement is technically feasible and appealing for children: in pediatric studies, urinary i-FABP was significantly higher in children with active disease than in controls, but discriminatory performance was only moderate, and levels in children already established on a gluten-free diet did not differ significantly from either group. Urinary sampling is therefore not yet validated against histology.

Plasma citrulline offers a complementary readout of enterocyte mass rather than immune activity. This non-protein amino acid is synthesized almost exclusively by mature enterocytes and converted to arginine mainly in the kidney, so its plasma concentration depends chiefly on intestinal production and renal clearance. Concentrations fall in proportion to villous atrophy severity and rise gradually over months of dietary treatment as the mucosa regenerates, closely paralleling histological recovery. Citrulline may help distinguish active villous atrophy from potential disease with preserved histology, though renal dysfunction, bowel resection and other enteropathies can confound results, and pediatric reference ranges remain unestablished.

On the immune side, cytokines capture the inflammatory response to gluten. Interleukin-2 rises within hours of gluten ingestion and is the best-characterized acute-response cytokine: multiplex studies have shown elevated serum IL-2 in the majority of treated patients after a single gluten challenge, with the magnitude of the rise correlating with symptom severity, and cytokine release after bolus challenge can discriminate celiac disease from self-reported non-celiac gluten sensitivity in patients already on a gluten-free diet. IL-8, IL-17A, IL-21, IL-6 and tumor necrosis factor-alpha are similarly elevated during active disease and generally correlate with antibody titers and villous atrophy severity. Clinical use, however, is constrained by short half-lives, inter-individual variability and the absence of standardized assays. High-mobility group box 1, an alarmin released from injured epithelium, is upregulated at diagnosis in children and appears to reflect histological damage severity, but it too is not disease-specific and pediatric evidence remains limited to small cohorts.

Stool and urine tests address a different problem: gluten exposure. Gluten immunogenic peptides are digestion-resistant gluten fragments detectable in stool for two to four days and in urine for 24 to 48 hours, providing objective evidence of recent intake, intentional or inadvertent, even when anti-TG2 antibodies are still normal. Fecal testing is more sensitive than dietary questionnaires for detecting poor adherence, though the short detection window means a negative result does not exclude intermittent lapses. The sensitivity of the method is underlined by a placebo-controlled dose-escalation study in treated adults in which urinary peptides became detectable in almost half of the patients after a single 50-milligram gluten dose, independently of symptoms. Current evidence supports these peptides primarily as adherence-monitoring tools rather than measures of mucosal healing.

Zonulin and fecal calprotectin are less convincing. Zonulin, a regulator of intestinal tight junctions, is elevated in serum and stool in newly diagnosed pediatric celiac disease, consistent with barrier dysfunction preceding villous atrophy, but commercial assays lack analytical specificity, recognizing properdin and other zonulin-related proteins rather than the pre-haptoglobin-2 precursor itself, and levels rise in numerous inflammatory and metabolic conditions. The review judges zonulin a research biomarker rather than a validated diagnostic tool. Fecal calprotectin, a marker of neutrophil-driven inflammation, is elevated in a proportion of untreated patients and typically declines after dietary treatment, but its correlation with histological severity is weak and it is raised across many gastrointestinal disorders.

Further out, microbiome and multi-omics research is generating molecular signatures that may eventually support risk prediction. Pediatric studies show intestinal dysbiosis in active disease, including reduced diversity, depletion of Bifidobacterium and Lactobacillus, and expansion of Bacteroides and Proteobacteria. Prospective birth-cohort metagenomic analyses in genetically at-risk infants have identified shifts in microbial strains, pathways and metabolites detectable up to 18 months before disease onset, and lipidomic studies have shown altered profiles months before gluten introduction in at-risk infants. In children with treated disease, fecal and urinary volatile organic compounds, short-chain fatty acids and free amino acids differed from controls despite at least two years on a gluten-free diet. RNA sequencing of duodenal biopsies has revealed immune, nutrient-transport and barrier gene-expression changes even in children whose first biopsy was non-diagnostic, and metabolomic profiling showed that most of 29 children with potential disease shared the metabolic profile of overt disease. None of these approaches is ready for routine clinical use.

The review’s bottom line is that no single biomarker is currently accurate and affordable enough to replace the ESPGHAN diagnostic algorithm, and the greatest benefit is likely to come from multimarker strategies combining serology with indicators of intestinal injury, immune activation and recent gluten exposure, particularly for patients with equivocal serology, selective IgA deficiency, potential or seronegative disease, or those already on a gluten-free diet. The authors note that this is a narrative rather than a systematic review, without formal risk-of-bias assessment, and that most emerging markers lack standardized assays, accepted reference ranges and prospective pediatric validation. Data from Central Asia are especially scarce, although a first prospective pediatric study from Uzbekistan identified celiac disease in 11 of 206 symptomatic children, findings that cannot be extrapolated to the general population. Before any of these tests reaches routine practice, the authors conclude, multicenter pediatric validation, assay standardization and age-specific reference ranges remain essential prerequisites.

Subject of Research: Emerging biomarkers for diagnosing and monitoring intestinal damage in pediatric celiac disease

Article Title: Biomarkers for intestinal damage in pediatric celiac disease

Article References: Sh. Usmonova, K., Geller, S. I., Umarnazarova, Z. E., & Kamilova, A. T. (2026). Biomarkers for intestinal damage in pediatric celiac disease. Frontiers in Pediatrics, 14, Article 2000459. https://doi.org/10.3389/fped.2026.2000459

Image Credits: AI Generated

DOI: 10.3389/fped.2026.2000459

Keywords: celiac disease, pediatrics, biomarkers, i-FABP, gluten immunogenic peptides, citrulline, zonulin, fecal calprotectin, microbiome, gluten-free diet, intestinal injury, multi-omics