Research Article: Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems

Date Published: January 14, 2019

Publisher: Public Library of Science

Author(s): Mary Ajamian, David Steer, Gennaro Rosella, Peter R. Gibson, Sabato D’Auria.


The protein, zonulin, has emerged as a popular serological marker to assess the integrity of the intestinal mucosal barrier. However, there is limited information on the utility of serum zonulin to indicate gastrointestinal disease and the validity of zonulin detection in widely-used commercial assays. The current study reports differences in zonulin levels across patient groups with gastrointestinal dysfunction compared with healthy individuals, though methodological inconsistencies indicated that actual zonulin protein was not detected by the commercial assays applied. The nature of the assays’ detected antigen was investigated using immunoprecipitation followed by mass spectrometric analysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein staining. Top matches of the assays’ detected antigen included haptoglobin and complement C3 for the assay manufactured by CUSABIO (Wuhan, China) and complement C3 for the assay manufactured by Immundiagnostik AG (Bensheim, Germany). These findings confirm that current commercial zonulin assays are not detecting the actual protein as prehaptoglobin-2. Until assay methodology is improved, we advise the greater scientific and medical community to exercise caution in considering the measurement of serum zonulin as a marker of mucosal barrier integrity.

Partial Text

Key roles of the intestinal epithelium are to regulate solute and fluid exchange as well as absorb nutrients [1]. An increasing number of studies point to an additional role of the gut epithelium and associated structures, along with gut-associated lymphoid tissue and the neuroendocrine network, as regulators in the passage of environmental antigens from the intestinal lumen into the sub-mucosa [2]. According to the proposed paradigm, dysregulation of the mucosal barrier leads to the increased passage of antigens and other macromolecules from the external environment into the host and initiates local and/or systemic inflammation and immune activation; this process attributed to “gut leakiness” influences tolerance and immunity.

Due to its putative role in reversible tight junction disassembly, circulating concentration of zonulin has emerged as an increasingly popular biological marker of mucosal barrier integrity. Despite its wide use to assess intestinal mucosal barrier integrity in clinical conditions where a “leaky gut” is suspected, there has been limited information on zonulin levels in patients with gastrointestinal dysfunction. In the current study, sera from well-characterised patient cohorts and controls were used to assess zonulin’s utility as a serological marker of gastrointestinal dysfunction and intestinal mucosal barrier integrity. However, the current commercial assays had significant methodological inconsistencies. Upon further investigation, the assays failed to detect recombinant zonulin/prehaptoglobin-2 protein.




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