Circulating Zonulin in Irritable Bowel Syndrome: A Systematic Review of Conflicting Evidence and the Critical Impact of Assay Specificity

Abstract

Background: Increased intestinal permeability is a proposed pathophysiological mechanism in irritable bowel syndrome (IBS), and zonulin, a putative regulator of tight junctions, has been investigated as a non-invasive serum biomarker. However, the diagnostic utility of circulating zonulin in IBS remains controversial. Objectives: To systematically review studies comparing serum or plasma zonulin concentrations between adult IBS patients and healthy controls, and to evaluate the influence of assay methodology on reported outcomes. Methods: A systematic literature search of PubMed was performed up to April 2026; the most recent eligible study was published in June 2024. Studies that enrolled adults with IBS diagnosed by Rome criteria, included healthy control groups, and measured zonulin by enzyme-linked immunosorbent assay (ELISA) were eligible. Studies lacking a healthy control group, non-original reports, animal studies, and mixed functional gastrointestinal disorder cohorts without extractable IBS data were excluded. Findings were synthesised narratively due to substantial methodological heterogeneity and concerns regarding the validity of commercially available zonulin kits. Risk of bias was assessed using the Newcastle–Ottawa Scale for case–control studies; six studies were at low risk and two at intermediate risk. The review is reported in accordance with the PRISMA 2020 statement, and this abstract follows the PRISMA 2020 for Abstracts checklist. Results: Eight case–control studies were included, comprising 421 IBS patients and 428 controls. Of the seven studies contributing to the primary synthesis, two reported significantly elevated zonulin in IBS, one produced opposite results with two different assays applied to the same samples, and four found no significant difference compared with controls. An eighth study, which combined IBS with inflammatory bowel disease and did not report IBS-specific data, was retained as supportive, non-primary evidence. Crucially, the studies employed different ELISA kits (ALPCO, Cusabio, Immundiagnostik, Zellbio) and different units of measurement. Independent validation studies have demonstrated that the most frequently used commercial kits do not recognise the pre-haptoglobin-2 molecule claimed to be zonulin, but instead detect haptoglobin, complement C3, or properdin. The only study employing a monoclonal antibody specific for pre-haptoglobin-2 observed lower, rather than higher, concentrations in IBS. Conclusions: The existing literature based on commercial ELISAs cannot determine whether pre-HP2/zonulin is altered in IBS, and the single genotype-controlled monoclonal assay suggests lower, not higher, concentrations in IBS. The disparate results are largely attributable to the application of ELISA kits that lack analytical specificity for the proposed target. Future research must use analytically validated assays, such as monoclonal antibody-based methods with concomitant genotyping for haptoglobin polymorphisms, before zonulin can be considered a meaningful biomarker in IBS. Funding: The Article Processing Charges were funded by the University of Medicine and Pharmacy of Craiova, Romania. Registration: The review was not prospectively registered; a retrospective protocol registration is available on OSF [10.17605/OSF.IO/MFA6J].