Noninvasive biomarkers of gut barrier function identify two subtypes of patients suffering from diarrhoea predominant-IBS: a case-control study

Michele Linsalata, Giuseppe Riezzo, Benedetta D'Attoma, Caterina Clemente, Antonella Orlando, Francesco Russo, Michele Linsalata, Giuseppe Riezzo, Benedetta D'Attoma, Caterina Clemente, Antonella Orlando, Francesco Russo

Abstract

Background: Alterations of the small-intestinal permeability (s-IP) might play an essential role in both diarrhoea-predominant IBS (D-IBS) and celiac disease (CD) patients. Our aims were to analyse in D-IBS patients the symptom profile along with the levels of urinary sucrose (Su), lactulose (La), mannitol (Ma), and circulating biomarkers (zonulin, intestinal fatty acid binding protein - I-FABP, and diamine oxidase - DAO) of the gastrointestinal (GI) barrier function. The pro-inflammatory interleukins 6 and 8 (IL-6 and IL-8), the plasma values of lipopolysaccharide (LPS), and Toll-like receptor 4 (TLR-4) were also investigated. Besides, these biomarkers were compared with those in CD and healthy controls (HC). Finally, comparisons were performed between D-IBS patients with [D-IBS(+)] and without [D-IBS(-)] increased s-IP according to normal or altered La/Ma ratio.

Methods: The study included 39 D-IBS patients, 32 CD patients, and 20 HC. GI permeability was assayed by high-performance liquid chromatography determination in the urine of Su and La/Ma ratio. ELISA kits assayed circulating concentrations of zonulin, I-FABP, DAO, IL-6, IL-8, LPS, and TLR-4. The Mann-Whitney or the Kruskal-Wallis with Dunn's post-test was used to assess differences among the groups.

Results: As for the La/Ma ratio, %Su, and I-FABP levels, D-IBS patients were significantly different from CD, but not HC. IL-6 levels were significantly higher in CD than HC, whereas IL-8 levels were significantly higher in both D-IBS and CD patients than HC. By opposite, LPS, and TLR-4 concentrations did not differ significantly among the groups. When D-IBS patients were categorised according to normal or altered s-IP, D-IBS(+) patients had %La, %Su, I-FABP, and DAO levels significantly higher than D-IBS(-) ones. The inflammatory parameters and markers of bacterial translocation (namely, IL-6 and LPS) were significantly higher in D-IBS(+) patients than D-IBS(-) ones.

Conclusions: The present study suggests that two distinct D-IBS subtypes could be identified. The investigation of possible s-IP alterations (i.e., considering the La/Ma ratio) might be useful to assess better and categorise this heterogeneous D-IBS population.

Trial registration: NCT01574209 . Registered March 2012. First recruitment started in April 2012.

Keywords: Celiac disease; Diarrhoea-predominant irritable bowel syndrome; Gut barrier; Interleukins; Intestinal permeability; Lipopolysaccharide.

Conflict of interest statement

Ethics approval and consent to participate

The study was approved by the Institutional Ethics Committee of IRCCS Ospedale Oncologico di Bari - Istituto Tumori Giovanni Paolo II, Bari, Italy, DDG reg. 1227/2013. The study was part of registered research on http://www.clinicaltrials.gov (reg. Number: NCT01574209). Written informed consent was obtained from all the patients and healthy participants for blood testing and clinical data collection.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
The flow of participants through the study D-IBS = diarrhoea-predominant IBS
Fig. 2
Fig. 2
%Ma, %La, La/Ma, and %Su in HC, D-IBS, and CD patients. A %Ma = Percentage of ingested mannitol recovered in urine. B %La = Percentage of ingested lactulose recovered in urine; C La/Ma = lactulose to mannitol ratio;  D %Su = Percentage of ingested sucrose recovered in urine. HC = Healthy controls. D-IBS = diarrhoea-predominant IBS. CD = celiac disease. Data are expressed as Mean ± SEM and analysed by Kruskal-Wallis test with Dunn’s Multiple Comparison Test. Means sharing the same superscript are not significantly different from each other (p < 0.05, Dunn’s test)
Fig. 3
Fig. 3
Serum Zonulin, I-FABP, and DAO levels in HC, D-IBS, and CD patients. A Zonulin; B I-FABP = Intestinal fatty acid binding protein; C DAO = diamine oxidase. HC = Healthy controls. D-IBS = diarrhoea-predominant IBS. CD = celiac disease. Data are expressed as Mean ± SEM and analysed by Kruskal-Wallis test with Dunn’s Multiple Comparison Test. Means sharing the same superscript are not significantly different from each other (p < 0.05, Dunn’s test)
Fig. 4
Fig. 4
Plasma concentrations of IL-6, IL-8, LPS, and TLR-4 in HC, D-IBS, and CD patients. A IL-6 = Interleukin-6;  B IL-8 = Interleukin-8; C LPS = Lipopolysaccharide; D TLR-4 = Toll-like receptor 4. HC = Healthy controls. D-IBS = diarrhoea-predominant IBS. CD = celiac disease. Data are expressed as Mean ± SEM and analysed by Kruskal-Wallis test with Dunn’s Multiple Comparison Test. Means sharing the same superscript are not significantly different from each other (p < 0.05, Dunn’s test)
Fig. 5
Fig. 5
Urinary markers of gastrointestinal barrier function in HC, D-IBS(−),D-IBS(+) patients, and CD patients HC = healthy controls. D-IBS = diarrhoea-predominant IBS. D-IBS patients with a Lactulose to Mannitol ratio lower than 0.035 were considered D-IBS(−). D-IBS patients with a ratio value equal to or higher than 0.035 were considered as D-IBS(+). CD = celiac disease. Urinary parameters of gastrointestinal permeability are expressed as percentages of ingested sugars recovered in urine: A mannitol (%Ma), B lactulose (%La), C the La/Ma ratio, and D sucrose (%Su). Data are expressed as Mean ± SEM and analysed by Kruskal-Wallis test with Dunn’s Multiple Comparison Test. Means sharing the same superscript are not significantly different from each other (p < 0.05, Dunn’s test)
Fig. 6
Fig. 6
Circulating markers of intestinal barrier function in HC, D-IBS(−), D-IBS(+) patients, and CD patients. HC = healthy controls. D-IBS = diarrhoea-predominant IBS. D-IBS patients with a Lactulose to Mannitol ratio lower than 0.035 were considered D-IBS(−). D-IBS patients with a ratio value equal to or higher than 0.035 were considered as D-IBS(+). CD = celiac disease. Circulating parameters of gastrointestinal permeability are expressed as: A Zonulin; B I-FABP = Intestinal fatty acid binding protein; C DAO = diamine oxidase. Data are expressed as Mean ± SEM and analysed by Kruskal-Wallis test with Dunn’s Multiple Comparison Test. Means sharing the same superscript are not significantly different from each other (p < 0.05, Dunn’s test)
Fig. 7
Fig. 7
Circulating levels of IL-6, IL-8, LPS, and TLR-4 in HC, D-IBS(−), D-IBS(+) patients, and CD patients. HC = healthy controls. D-IBS = diarrhoea-predominant IBS. D-IBS patients with a Lactulose to Mannitol ratio lower than 0.035 were considered D-IBS(−). D-IBS patients with a ratio value equal to or higher than 0.035 were considered as D-IBS(+). CD = celiac disease. A IL-6 = Interleukin-6; B IL-8 = Interleukin-8; C LPS = Lipopolysaccharide; D TLR-4 = Toll-like receptor 4. Data are expressed as Mean ± SEM and analysed by Kruskal-Wallis test with Dunn’s Multiple Comparison Test. Means sharing the same superscript are not significantly different from each other (p < 0.05, Dunn’s test)

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