Review for 10.1186/s12876-021-02075-y (2022)

Title: The association of zonulin-related proteins withprevalent and incident inflammatory
bowel disease
Version: 2 Date: 22 Oct 2021
Reviewer's report:
In the present manuscript, Wang and colleagues investigated serum zonulin levels in patients
with IBD vs matched controls, and the association between serum zonulin levels and the
development of IBD in subject on long term FU.
Among a large cohort of subject included in WHILA study, those with a baseline diagnosis
of IBD (n=18) showed higher serum zonulin level compared to controls; however, the study
failed to demonstrate an association between baseline serum zonulin values and the incidence
of IBD, following a matched analysis between subjects that developed IBD while on FU (n=56)
vs matched subject that did not develop the disease.
Considering that zonulin has been proposed as biomarker of intestinal permeability, in this
regard, the results of the study support the recent evidences of an impaired intestinal permeability
in patients with IBD compared to those without; however, given the lack of statistical
significance in the comparison between incident cases and matched controls, it seems that
impaired intestinal permeability could be a consequence, rather that a cause of the chronic
inflammatory response that characterizes both CD and UC.
Overall, the study is interesting and the cohort analyzed represents a great opportunity to assess
the the role of intestinal permeability (measured by zonulin, with the limitations of point 2) in
the development of IBD.
Below the specific comments.
1) Introduction. There are some bachground aspects that need to amended; in particular: "UC
involves ulcers formed in the colon and rectum"; ulcers characterized more severe forms of
UC. I suggest to amend with a more general "inflammation". "CD is more common than UC
but with similar symptoms"; diarrhea and bleeding are more common in UC, while in CD
is more common abdominal pain and weight loss. "Prevalence ranges from 10-200 cases per
100,000 individuals"; in Western Countries, prevalence is a bit higher, between 200-400 cases
per 100,000 individuals.
2) Methods. Serum zonulin was measured by a commercially available ELISA method
(Immunodiagnostiks). Recent studies raised important concerns regarding the specificity of the
two more widely adopted commercial assays for the measurement of zonulin (see Ajamian
M et al. Serum zonulin as a marker of intestinal mucosal barrier function: May not be what
it seems. PLoS One 2019 and Scheffler L et al. Widely Used Commercial ELISA Does Not
Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member
of the Zonulin Family. Front Endocrinol 2018.), and consequently the reliability of zonulin as
marker of intestinal permeability has been strongly challenged. Therefore, I suggest to add this
aspect in the discussion of the manuscript. Furthermore, as explained by Fasano (Gut 2020)
and as indicated on the manual of the IDK Zonulin Assay, zonulin is not exclusively pre-
aptoglobin 2, rather is a family of structurally and functionally related proteins; in this regard, I
recommend the authors to amend "zonulin" with "zonulin related proteins (ZRP)"  throughout
the whole manuscript. These aspects should be highlighted in the introduction section, and their
implications should be discussed in the discussion section.