Gut permeability marker zonulin in sickle cell disease: Preliminary associations with vaso-occlusive crisis frequency

Abstract Background: Vaso-occlusive crises (VOC) are a hallmark complication of sickle cell disease (SCD), driven by neutrophil activation and vascular inflammation. Emerging research implicates gut dysbiosis and epithelial barrier dysfunction as potential contributors to VOC pathophysiology, possibly through systemic translocation of pro-inflammatory bacterial metabolites. Zonulin, a biomarker of intestinal permeability and a key player in maintaining a healthy gut barrier, may reflect this translocation risk. However, data linking zonulin to VOC burden in SCD patients is lacking. The objective of this study was to evaluate whether stool zonulin concentrations, plasma zonulin concentrations, and the plasma-to-stool zonulin ratio differ between sickle cell disease patients with frequent vaso-occlusive crises (cases) and those with infrequent crises (controls). Methods: This is an ongoing prospective, observational cohort study of adults with sickle cell disease, conducted over an 18-month period. Participants were stratified into two cohorts based on vaso-occlusive crisis (VOC) frequency in the prior 12 months: a control group with two or fewer VOCs and a case group with three or more VOCs. To assess differences in zonulin concentrations by group, all available plasma samples and all available stool samples were analyzed separately to compare average levels between cases and controls. In addition, for participants who provided both plasma and stool samples, a plasma-to-stool zonulin ratio was calculated to allow for a matched-pair comparison between the two groups. Blood samples were collected during routine clinic visits, and stool samples were self-collected by participants and mailed to our laboratory, where they were processed upon arrival. Zonulin (Zonulin Family Peptides) quantification was performed by Enzyme-Linked Immunosorbent Assay (ELISA) using two Immunodiagnostik kits specific to stool or serum samples, following the manufacturer's guidelines. Stool samples were prepared using the stool sample application system from Immunodiagnostik. Group differences in plasma zonulin, stool zonulin, and plasma-to-stool ratio between cases and controls were evaluated using the Mann–Whitney U test due to non-normal data distribution. Results were expressed as medians and interquartile ranges (IQRs), and a two-sided p-value of less than 0.05 was considered statistically significant. Statistical analyses were performed using R (R Foundation for Statistical Computing, Vienna, Austria) and Stata (StataCorp, College Station, TX) version 18. Results: A total of 38 plasma samples, 20 stool samples, and 15 matched plasma-to-stool ratio values were included in the analysis. For plasma, the median zonulin level in the case group was 10.57 ng/mL (IQR 9.61–12.24), compared to 10.31 ng/mL (IQR 9.31–12.09) in the control group (p = 0.437). For stool, the median zonulin level in cases was 43.06 ng/mL (IQR 31.06–63.70), compared to 48.12 ng/mL (IQR 33.98–65.66) in controls (p = 0.850). The plasma-to-stool ratio was higher in cases, with a median of 0.34 (IQR 0.19–0.42) versus 0.16 (IQR 0.11–0.50) in controls (p = 0.336). Conclusions: Although not statistically significant, the case group demonstrated slightly higher plasma zonulin concentrations and plasma-to-stool ratios compared to controls, suggesting a possible trend toward increased systemic zonulin exposure relative to intestinal levels. Interestingly, stool zonulin levels were slightly lower in the case cohort, which may reflect altered compartmentalization or regulation of zonulin in patients with more frequent VOCs. These findings support the need for larger, longitudinal studies to explore whether gut barrier alterations contribute to VOC risk and to clarify the mechanistic relationship between intestinal permeability biomarkers, vascular inflammation, and VOC risk in SCD.