Blurring the picture in leaky gut research: how ...

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Table 1 Studies using zonulin ELISA and correlations with intestinal permeability

Study Year Zonulin kit N Correlation Citation

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

Kuzma et al 2020 IDK(distributed by ALPCO) 24 R=0.033, p=0.79 10

Blurring the picture in leaky gut research: how shortcomings of zonulin as a biomarker mislead the field of intestinal permeability

With great interest we read the work by Talley et al1 reporting the inadequacy of zonulin as a biomarker due to its failure to identify the irritable bowel syndrome, functional dyspepsia and non- coeliac wheat sensitivity. Zonulin as a biomarker is highly disputed.2 A recent study showed that zonulin- mediated intestinal barrier integrity is an important mechanism by which gut microbial dysbiosis affects the transition from asymptotic autoimmunity to inflammatory disease associated with increased circulating zonulin in patients with arthritis.3 In all of these studies, zonulin measurements are based on commercial ELISA.

There is no doubt about the clinical relevance of studies addressing the rela-tion between intestinal permeability and inflammatory diseases. Zonulin, precisely pre- haptoglobin 2 (preHP2), was identi-fied as a human homologue to a second Vibrio cholerae enterotoxin regulating tight junction permeability and subse-quently has gained much attention as a potential biomarker for intestinal perme-ability.4 However, the commercial ELISAs very frequently used to measure zonulin were produced using the first published sequence, which later has been shown to be unrelated to the zonulin protein.4 These developments have resulted in the following two major critical yet widely overlooked issues.

COMMERCIALLY AVAILABLE ELISAS DO NOT MEASURE ZONULINThe shortcomings of the commercial ELISA have been demonstrated in inde-pendent work and have been discussed previously.5 6 Measurements using these commercial ELISA do not reflect actual zonulin levels, but concentrations of unknown proteins. Consequently, this has to preclude scientists from drawing conclusions on the role and importance of zonulin in the context of intestinal

permeability and related diseases based on these ELISA measurements, both positive and negative. This, also retrospectively, applies to numerous studies reporting findings relying on the commercial ELISA kits.6 Furthermore, these zonulin ELISA measurements only poorly correlate with functional gut permeability as assessed by, for example, lactulose mannitol test (table 1).

Importantly, this does not take away from zonulin/preHP2 as a regulator of intestinal permeability and does not rule out correlations of zonulin levels with intestinal barrier function.

ZONULIN AS PRE-HAPTOGLOBIN2 IS NOT EXPRESSED IN MICEAnimal models of intestinal barrier dysfunction are highly useful for trans-lational research, yet zonulin as preHP2 is not naturally expressed in mice. While haptoglobin is conserved in most mammals, the HP2 genotype is unique to humans. This renders measurements of serum zonulin in rodent models highly questionable and potentially misleading.3 Along these lines, differential ELISA signals obtained in mouse sera further indi-cate detection of unspecific and unknown proteins by the ELISA.3 For translational research, assessing zonulin levels in mouse models does only become relevant when using zonulin- specific assays in ‘human-ised mice’ genetically modified to express human HP2, as has been previously described.7

CONCLUSIONTogether, it has become obvious that using the commercial zonulin ELISA is neither adequate to measure intestinal perme-ability nor the postulated biomarker zonulin. Even more important, previously published results based on zonulin ELISA measurements have to be seen with great caution and do not establish a relation to the function of the protein zonulin/preHP2. New and specific detection methods and assays for zonulin/preHP2 are urgently needed to address the useful-ness of zonulin as a biomarker for intes-tinal permeability. Until then, researchers are strongly encouraged to circumvent the unspecific measurement of zonulin and

instead apply rigorous tests of intestinal permeability such as dual- sugar assays, and use immunohistochemistry and expression profiles of zonula occludens proteins.3

Lucas Massier,1 Rima Chakaroun,1 Peter Kovacs ,1 John T. Heiker2

1Medical Department III – Endocrinology, Nephrology, Rheumatology, University of Leipzig Medical Center, Leipzig, Saxony, Germany2Helmholtz Institute for Metabolic, Obesity and Vascular Research (HI- MAG) of the Helmholtz Zentrum München at Leipzig University and University Hospital Leipzig, Leipzig, Saxony, Germany

Correspondence to Dr Peter Kovacs, Medical Department III – Endocrinology, Nephrology, Rheumatology, University of Leipzig Faculty of Medicine, Leipzig, Sachsen, Germany; Peter. Kovacs@ medizin. uni- leipzig. de and Dr John T. Heiker, Helmholtz Institute for Metabolic, Obesity and Vascular Research (HI- MAG) of the Helmholtz Zentrum München at Leipzig University and University Hospital Leipzig, Leipzig, Germany; john. heiker@ helmholtz- muenchen. de

Contributors All authors contributed equally in writing and editing of the letter.

Funding Lucas Massier was funded and this work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation – Projektnummer 209933838 – SFB 1052/B03, SFB 1052/C07).

Competing interests None declared.

Patient consent for publication Not required.

Provenance and peer review Not commissioned; internally peer reviewed.

Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY- NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non- commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non- commercial. See: http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.

© Author(s) (or their employer(s)) 2021. Re- use permitted under CC BY- NC. No commercial re- use. See rights and permissions. Published by BMJ.

LM, RC, PK and JTH contributed equally.

To cite Massier L, Chakaroun R, Kovacs P, et al. Gut 2021;70:1801–1802.

Received 8 September 2020Revised 27 September 2020Accepted 30 September 2020Published Online First 9 October 2020

Gut 2021;70:1801–1802. doi:10.1136/gutjnl-2020-323026

ORCID iDPeter Kovacs http:// orcid. org/ 0000- 0002- 0290- 5423

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REFERENCES 1 Talley NJ, Holtmann GJ, Jones M, et al. Zonulin

in serum as a biomarker fails to identify the IBS, functional dyspepsia and non- coeliac wheat sensitivity. Gut 2020;69:1–3.

2 Barbaro MR, Cremon C, Morselli- Labate AM, et al. Serum zonulin and its diagnostic performance in non- coeliac gluten sensitivity. Gut 2020;69:1966–74.

3 Tajik N, Frech M, Schulz O, et al. Targeting zonulin and intestinal epithelial barrier function to prevent onset of arthritis. Nat Commun 2020;11:1–14.

4 Tripathi A, Lammers KM, Goldblum S, et al. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proc Natl Acad Sci U S A 2009;106:16799–804.

5 Scheffler L, Crane A, Heyne H, 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;9:22.

6 Ajamian M, Steer D, Rosella G, et al. Serum zonulin as a marker of intestinal mucosal barrier function: may not be what it seems. PLoS One 2019;14:e0210728.

7 Miranda- Ribera A, Ennamorati M, Serena G, et al. Exploiting the zonulin mouse model to establish the role of primary impaired gut barrier function on microbiota composition and immune profiles. Front Immunol 2019;10:2233.

8 Hałasa M, Maciejewska D, Ryterska K, et al. Assessing the association of elevated zonulin concentration in stool with increased intestinal permeability in active professional athletes. Medicina 2019;55:710.

9 Linsalata M, Riezzo G, D’Attoma B, et al. Noninvasive biomarkers of gut barrier function identify two subtypes of patients suffering from diarrhoea predominant- IBS: a case- control study. BMC Gastroenterol 2018;18:167.

10 Kuzma JN, Hagman DK, Cromer G, et al. Intraindividual variation in markers of intestinal permeability and adipose tissue inflammation in healthy normal- weight to obese adults. Cancer Epidemiol Biomarkers Prev 2019;28:610–5.

A nationwide cohort study with propensity score matching

Lee and colleagues recently published the first large- scale study to investigate the association between proton pump inhib-itor (PPI) use and the infectious disease caused by COVID-19.1 Using a nation-wide cohort sample with propensity score matching, they concluded that short- term current—but neither long- term current nor past—PPI usage was associated with worse outcomes of COVID-19. These results deserve some comments.

By decreasing the barrier effect of gastric acidity and thus promoting the survival of ingested pathogens, PPIs are a known risk factor for some enteric bacterial and virus infections.2 3 Based on the evidence for a fecal–oral transmission in COVID-19, the authors made the hypothesis that PPI use might influence the susceptibility to COVID-19. Nevertheless, criteria other than biological plausibility should be taken into account when considering retrospective observational studies, in which information is not collected with a

specific hypothesis in mind; level of expo-sure to PPIs is unknown; and confounders and biases persist even after adjustments.4

One of these criteria is strength of association. In the study by Lee and colleagues, all adjusted ORs are below 2. Because weaker associations are less likely to be causal, some authors recommend that results of risk estimates (OR or RR) between 0.5 and 2.0 (also referred to as the ‘zone of potential bias’) should be rejected and considered non- informative.5 Applying this criterion to the present study leads to questioning the clinical relevance of its findings.

In their discussion, Lee and colleagues reported that their study accounted for protopathic bias by excluding new nonste-roidal anti- inflammatory drugs users and designing propensity score matching. However, their results are very sugges-tive of protopathic bias since the increase in risk of worse clinical outcomes of COVID-19 only occurred in patients newly exposed to PPIs, this risk disap-pearing in patients exposed for 1 month or more. It can therefore be hypothesised that a PPI was introduced in some of these patients in response to the early diges-tive symptoms of COVID-19, before the infection was diagnosed. As noted by the authors, the same concerns about proto-pathic bias have been raised about the association between PPI use and risk of pneumonia.6

Lastly, a statistically significant asso-ciation was found between PPI use and worse outcomes of COVID-19, but not between PPI use and the infection rates of COVID-19 among tested patients, which suggests that confounding by indication seems very likely. Stress ulcer prophylaxis is actually recommended to be administered to critically ill patients who are assessed as high risk for GI bleeding, including those requiring mechanical ventilation or high- dose corticosteroids.7 Given the criteria used to construct the composite endpoints 1 and 2 (ie, requirement of oxygen therapy, intensive care unit admission, administration of invasive ventilation, severe clinical outcomes of COVID-19 or death), the study was designed to select patients with both PPI prescription and worse outcomes of COVID-19. Baseline characteristics of included patients (see table 1) support this hypothesis, with patients in the ‘current PPI use group’ being older and having more comorbidi-ties than in the other groups. The use of propensity score matching was a valuable but probably insufficient effort to fully balance these major differences in baseline characteristics.

For all these reasons, these results should be interpreted with caution. In the patients most severely affected by COVID-19 who require intensive care management, the proven benefits of PPIs should not be outweighed by a risk that remains hypothetical to date.

Lucien Roulet 1

Department of Clinical Pharmacy, Hospital Pharmacy of Nord Vaudois and Broye Region, Yverdon- les- Bains, Switzerland

Correspondence to Dr Lucien Roulet, Clinical pharmacy, Association de la pharmacie centrale des Hôpitaux du Nord Vaudois et de la Broye, Yverdon- les- Bains, Switzerland; lucien. roulet@ gmail. com

Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not- for- profit sectors.

Competing interests None declared.

Patient and public involvement Patients and/or the public were not involved in the design, conduct, reporting or dissemination plans of this research.

Patient consent for publication Not required.

Provenance and peer review Not commissioned; internally peer reviewed.

This article is made freely available for use in accordance with BMJ’s website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by BMJ. You may use, download and print the article for any lawful, non- commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained.

© Author(s) (or their employer(s)) 2021. No commercial re- use. See rights and permissions. Published by BMJ.

To cite Roulet L. Gut 2021;70:1802–1803.

Received 15 September 2020Accepted 8 October 2020Published Online First 19 October 2020

Gut 2021;70:1802–1803. doi:10.1136/gutjnl-2020-323098

ORCID iDLucien Roulet http:// orcid. org/ 0000- 0001- 9421- 7742

REFERENCES 1 Lee SW, Ha EK, Yeniova Abdullah Özgür, Yeniova AÖ,

et al. Severe clinical outcomes of COVID-19 associated with proton pump inhibitors: a nationwide cohort study with propensity score matching. Gut 2021;70:76–84.

2 Hafiz RA, Wong C, Paynter S, et al. The risk of community- acquired enteric infection in proton pump inhibitor therapy: systematic review and meta- analysis. Ann Pharmacother 2018;52:613–22.

3 Charpiat B, Bleyzac N, Tod M. Proton pump inhibitors are risk factors for viral infections: even for COVID-19? Clin Drug Investig 2020:1–3.

4 Schubert ML. Adverse effects of proton pump inhibitors: fact or fake news? Curr Opin Gastroenterol 2018;34:451–7.

5 Grimes DA, Schulz KF. False alarms and pseudo- epidemics: the limitations of observational epidemiology. Obstet Gynecol 2012;120:920–7.

6 Wang C- H, Li C- H, Hsieh R, et al. Proton pump inhibitors therapy and the risk of pneumonia: a systematic review and meta- analysis of randomized controlled

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