Letter
Blurring the picture in leaky gut Table 1 Studies using zonulin ELISA and correlations with intestinal permeability Gut: first published as 10.1136/gutjnl-2020-323026 on 9 October 2020. Downloaded from research: how shortcomings of Study Year Zonulin kit N Correlation Citation zonulin as a biomarker mislead Halasa et al 2019 IDK 38 R=0.11, p>0.05 8 Linsalata et al 2018 IDK 71 R=0.17, p>0.05 9 the field of (distributed by ALPCO) 10 intestinal permeability Kuzma et al 2020 IDK 24 R=0.033, p=0.79
With great interest we read the work by 1 by, for example, lactulose mannitol test Medical Department III – Endocrinology, Nephrology, Talley et al1 reporting the inadequacy of (table 1). Rheumatology, University of Leipzig Medical Center, zonulin as a biomarker due to its failure Leipzig, Saxony, Germany Importantly, this does not take away 2 to identify the irritable bowel syndrome, Helmholtz Institute for Metabolic, Obesity and Vascular from zonulin/preHP2 as a regulator of functional dyspepsia and non-coeliac Research (HI-MAG) of the Helmholtz Zentrum München intestinal permeability and does not rule at Leipzig University and University Hospital Leipzig, wheat sensitivity. Zonulin as a biomarker out correlations of zonulin levels with Leipzig, Saxony, Germany is highly disputed.2 A recent study showed intestinal barrier function. that zonulin-mediated intestinal barrier Correspondence to Dr Peter Kovacs, Medical Department III – Endocrinology, Nephrology, integrity is an important mechanism by Rheumatology, University of Leipzig Faculty of which gut microbial dysbiosis affects the ZONULIN AS PRE-HAPTOGLOBIN2 IS NOT Medicine, Leipzig, Sachsen, Germany; transition from asymptotic autoimmunity EXPRESSED IN MICE Peter. Kovacs@medizin. uni-leipzig. de and Dr John T. to inflammatory disease associated with Heiker, Helmholtz Institute for Metabolic, Obesity and Animal models of intestinal barrier Vascular Research (HI-MAG) of the Helmholtz Zentrum increased circulating zonulin in patients 3 dysfunction are highly useful for trans- München at Leipzig University and University Hospital with arthritis. In all of these studies, lational research, yet zonulin as preHP2 Leipzig, Leipzig, Germany; john. heiker@helmholtz- zonulin measurements are based on is not naturally expressed in mice. muenchen.de commercial ELISA. While haptoglobin is conserved in most Contributors All authors contributed equally in There is no doubt about the clinical mammals, the HP2 genotype is unique writing and editing of the letter. relevance of studies addressing the rela- to humans. This renders measurements Funding Lucas Massier was funded and this tion between intestinal permeability and of serum zonulin in rodent models highly work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG, German Research inflammatory diseases. Zonulin, precisely questionable and potentially misleading.3 pre-haptoglobin 2 (preHP2), was identi- Foundation – Projektnummer 209933838 – SFB 1052/ Along these lines, differential ELISA B03, SFB 1052/C07). fied as a human homologue to a second signals obtained in mouse sera further indi- Competing interests None declared. Vibrio cholerae enterotoxin regulating cate detection of unspecific and unknown Patient consent for publication Not required. tight junction permeability and subse- proteins by the ELISA.3 For translational quently has gained much attention as a research, assessing zonulin levels in mouse Provenance and peer review Not commissioned; potential biomarker for intestinal perme- internally peer reviewed. 4 models does only become relevant when http://gut.bmj.com/ ability. However, the commercial ELISAs using zonulin-specific assays in ‘human- very frequently used to measure zonulin ised mice’ genetically modified to express were produced using the first published human HP2, as has been previously sequence, which later has been shown described.7 to be unrelated to the zonulin protein.4 These developments have resulted in the Open access This is an open access article
following two major critical yet widely CONCLUSION distributed in accordance with the Creative Commons on September 28, 2021 by guest. Protected copyright. overlooked issues. Together, it has become obvious that using Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build the commercial zonulin ELISA is neither upon this work non-commercially, and license their adequate to measure intestinal perme- COMMERCIALLY AVAILABLE ELISAS DO derivative works on different terms, provided the ability nor the postulated biomarker original work is properly cited, appropriate credit is NOT MEASURE ZONULIN zonulin. Even more important, previously given, any changes made indicated, and the use is The shortcomings of the commercial published results based on zonulin ELISA non-commercial. See: http://creativecommons.org/ ELISA have been demonstrated in inde- licenses/by-nc/4.0/. measurements have to be seen with great pendent work and have been discussed caution and do not establish a relation © Author(s) (or their employer(s)) 2020. Re-use previously.5 6 Measurements using these permitted under CC BY-NC. No commercial re-use. See to the function of the protein zonulin/ commercial ELISA do not reflect actual rights and permissions. Published by BMJ. preHP2. New and specific detection zonulin levels, but concentrations of LM, RC, PK and JTH contributed equally. methods and assays for zonulin/preHP2 unknown proteins. Consequently, this are urgently needed to address the useful- has to preclude scientists from drawing ness of zonulin as a biomarker for intes- conclusions on the role and importance tinal permeability. Until then, researchers To cite Massier L, Chakaroun R, Kovacs P, et al. Gut of zonulin in the context of intestinal are strongly encouraged to circumvent the Epub ahead of print: [please include Day Month Year]. permeability and related diseases based on unspecific measurement of zonulin and doi:10.1136/gutjnl-2020-323026 these ELISA measurements, both positive instead apply rigorous tests of intestinal Received 8 September 2020 and negative. This, also retrospectively, permeability such as dual-sugar assays, and Revised 27 September 2020 applies to numerous studies reporting Accepted 30 September 2020 use immunohistochemistry and expression findings relying on the commercial ELISA 3 kits.6 Furthermore, these zonulin ELISA profiles of zonula occludens proteins. Gut 2020;0:1–2. doi:10.1136/gutjnl-2020-323026 measurements only poorly correlate with Lucas Massier,1 Rima Chakaroun,1 ORCID iD functional gut permeability as assessed Peter Kovacs ,1 John T. Heiker2 Peter Kovacs http://orcid. org/0000- 0002-0290- 5423
Gut Month 2020 Vol 0 No 0 1 Letter
REFERENCES modulator of tight junctions, as prehaptoglobin-2. Proc 8 Hałasa M, Maciejewska D, Ryterska K, et al. Natl Acad Sci U S A 2009;106:16799–804. Assessing the association of elevated zonulin
1 Talley NJ, Holtmann GJ, Jones M, et al. Zonulin Gut: first published as 10.1136/gutjnl-2020-323026 on 9 October 2020. Downloaded from in serum as a biomarker fails to identify the IBS, 5 Scheffler L, Crane A, Heyne H, et al. Widely used concentration in stool with increased intestinal functional dyspepsia and non-coeliac wheat sensitivity. commercial ELISA does not detect precursor of permeability in active professional athletes. Medicina Haptoglobin2, but recognizes properdin as a potential 2019;55:710. Gut 2020;69:1–3. second member of the zonulin family. Front Endocrinol 9 Linsalata M, Riezzo G, D’Attoma B, et al. Noninvasive 2 Barbaro MR, Cremon C, Morselli-Labate AM, 2018;9:22. biomarkers of gut barrier function identify two et al. Serum zonulin and its diagnostic 6 Ajamian M, Steer D, Rosella G, et al. Serum zonulin as subtypes of patients suffering from diarrhoea performance in non-coeliac gluten sensitivity. Gut a marker of intestinal mucosal barrier function: may predominant-IBS: a case-control study. BMC 2020;69:1966–74. not be what it seems. PLoS One 2019;14:e0210728. Gastroenterol 2018;18:167. 3 Tajik N, Frech M, Schulz O, et al. Targeting zonulin and 7 Miranda-Ribera A, Ennamorati M, Serena G, et al. 10 Kuzma JN, Hagman DK, Cromer G, et al. Intraindividual intestinal epithelial barrier function to prevent onset of Exploiting the zonulin mouse model to establish variation in markers of intestinal permeability and arthritis. Nat Commun 2020;11:1–14. the role of primary impaired gut barrier function on adipose tissue inflammation in healthy normal-weight 4 Tripathi A, Lammers KM, Goldblum S, et al. microbiota composition and immune profiles. Front to obese adults. Cancer Epidemiol Biomarkers Prev Identification of human zonulin, a physiological Immunol 2019;10:2233. 2019;28:610–5. http://gut.bmj.com/ on September 28, 2021 by guest. Protected copyright.
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