Ulcerative Colitis Permeability

Abstract: This cross-sectional, exploratory proof-of-concept study aims to investigate whether serum zonulin and lipopolysaccharide-binding protein (LBP) levels, biomarkers of intestinal permeability, are associated with the severity of hepatic steatosis and fibrosis in adult patients with ulcerative colitis (UC) and metabolic dysfunction-associated steatotic liver disease (MASLD). The study was intentionally designed as a proof-of-concept investigation; therefore, a relatively small sample size (approximately 100 participants, with 82 required according to the power analysis) was selected a priori to explore potential associations and generate hypotheses for future research.

Hypothesis: Among patients with UC and MASLD, higher serum concentrations of zonulin and/or LBP are expected to be associated with increased odds of significant liver fibrosis, defined as liver stiffness measurement (LSM) >8 kPa. Similar associations are anticipated for hepatic steatosis, defined by a controlled attenuation parameter (CAP) ≥274 dB/m, and for advanced fibrosis (LSM >12 kPa). Because the distribution of LSM and CAP values in this population is unknown, additional multiple linear regression analyses will evaluate these outcomes as continuous variables to explore the strength and direction of associations across their full spectrum.

Study Population and Eligibility Criteria: Approximately 100 adult patients with UC in both clinical remission (partial Mayo score <2) and biochemical remission (fecal calprotectin <100 μg/g) will be recruited from a single tertiary-care center. Inclusion will require reliable transient elastography measurements obtained using FibroScan.

Major exclusion criteria include excessive alcohol consumption; previous liver diseases of autoimmune, viral, alcoholic, or hereditary/metabolic etiology; decompensated cirrhosis; celiac disease; type 1 diabetes mellitus; hepatitis B or C infection; previous malignancy; marked transaminase elevation (>5 times the upper limit of normal); hepatic congestion; biliary obstruction; portal vein thrombosis; Budd-Chiari syndrome; pregnancy; age below 18 years; inability or refusal to provide informed consent; and ulcerative colitis limited to the rectum (ulcerative proctitis).

A sample size of 82 participants provides 80% statistical power to detect a clinically relevant difference in MASLD prevalence between groups with high and low biomarker levels. To account for missing data and potential exclusions, approximately 100 participants will be enrolled.

Methods and Statistical Analysis: All participants will undergo transient elastography to assess CAP and LSM values, together with blood sampling for measurement of serum zonulin and LBP concentrations using enzyme-linked immunosorbent assay (ELISA).

Demographic variables (age, sex), anthropometric parameters (body mass index [BMI]), inflammatory markers (C-reactive protein and fecal calprotectin), and current UC-related therapy will be recorded.

Data distribution will be assessed using appropriate normality tests. Continuous variables will be summarized as mean ± standard deviation or median with interquartile range, depending on distribution. Group comparisons will be performed using Student's t-test or the Mann-Whitney U test, while correlations will be assessed using Spearman's rank correlation coefficient.

Multivariable logistic regression models will evaluate whether higher zonulin and/or LBP concentrations are independently associated with significant fibrosis (LSM >8 kPa), advanced fibrosis (LSM >12 kPa), and hepatic steatosis (CAP ≥274 dB/m), after adjustment for potential confounders including BMI, age, sex, C-reactive protein levels, and current therapy.

Separate multivariable linear regression models will analyze continuous CAP and LSM values using the same covariates and will additionally explore potential interaction effects. Prior to complete-case analysis, the extent and pattern of missing data will be evaluated descriptively to assess the risk of selection bias.

Expected Outcomes and Significance: By clarifying whether biomarkers of intestinal permeability are independently associated with hepatic steatosis and fibrosis in patients with UC and MASLD, this study seeks to determine whether zonulin and LBP may serve as early indicators of liver injury in this population.

Positive findings would support the biological relevance of the gut-liver axis in MASLD and provide justification for larger prospective longitudinal studies incorporating repeated measurements, mechanistic investigations, and external validation cohorts. Conversely, the absence of significant associations would suggest that these biomarkers have limited utility as indicators of liver injury in patients with UC and MASLD.

Given the cross-sectional design, the study is intended to identify associations rather than establish causal relationships.

Inclusion criteria

• Adults aged 18-70 years.
• Established diagnosis of ulcerative colitis (UC) for at least 6 months according to current European Crohn's and Colitis Organisation (ECCO) guidelines.
• Clinical remission of UC defined as a partial Mayo score <2.
• Biochemical remission of UC defined as fecal calprotectin (FC) <100 μg/g.
• Ability to undergo transient elastography (FibroScan) with reliable measurements.
• Written informed consent provided prior to study participation.

Exclusion criteria

• Ulcerative colitis limited to the rectum (ulcerative proctitis) according to the most recent endoscopic assessment.
• Excessive alcohol consumption (≥140 g/week for women or ≥210 g/week for men).
• Unreliable transient elastography measurements (CAP or LSM).
• Known chronic liver disease of other etiology, including autoimmune, viral, alcoholic, metabolic, or malignant liver disease.
• Decompensated liver cirrhosis.
• Positive hepatitis B virus (HBV) or hepatitis C virus (HCV) serology.
• Celiac disease.
• Type 1 diabetes mellitus.
• History of malignancy.
• Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels >5 times the upper limit of normal.
• Hepatic congestion due to heart failure.
• Biliary obstruction with cholestatic liver enzyme abnormalities.
• Neoplastic liver infiltration.
• Portal vein thrombosis.
• Budd-Chiari syndrome.
• Pregnancy.
• Inability or unwillingness to provide written informed consent.