Biomarkers of Gut Epithelia Integrity in the ENDOTARGET project

What are Biomarkers?

Biomarkers are measurable indicators of biological processes, health conditions, or responses to treatment. They can be molecules found in blood, tissues, or other body fluids (e.g., proteins, genes, metabolic byproducts) or even physiological measures like blood pressure.

Biomarkers play a crucial role in diagnosing diseases, predicting risks, monitoring treatment effectiveness, and understanding disease mechanisms. By identifying and studying biomarkers, more precise and personalised medical approaches can be developed.

 

Biomarkers of Gut Epithelia Integrity

The gut epithelium is a crucial barrier that regulates the exchange of nutrients while preventing harmful substances, such as bacteria and toxins, from entering the bloodstream. When this barrier is compromised, often referred to as “leaky gut” or increased intestinal permeability, it can contribute to inflammation, endotoxemia, and autoimmune diseases.

To assess gut epithelial integrity, scientists and clinicians rely on specific biomarkers that indicate barrier function, permeability, and damage.

1. Zonulin

Zonulin is a regulatory protein that controls the tight junctions between cells in the intestinal epithelium. These tight junctions act as gates, selectively allowing nutrients and water to pass while preventing toxins, pathogens, and undigested food particles from entering the bloodstream. When zonulin levels increase, tight junctions loosen, leading to increased intestinal permeability. Thus, zonulin is an important biomarker for intestinal permeability.

Zonulin is released in response to triggers like gluten, bacterial overgrowth, or infections. It temporarily increases gut permeability to help with immune surveillance, but excessive or prolonged zonulin activation can weaken the intestinal barrier, allowing harmful substances to enter the bloodstream. This can contribute to chronic inflammation, endotoxemia, and immune dysregulation, which are linked to various autoimmune and inflammatory diseases.

Zonulin levels can be measured in blood serum, reflecting systemic zonulin levels and potential gut permeability, and in faeces, indicating zonulin production within the intestines, often used in gut health assessments.

Elevated Zonulin levels are associated with certain diseases, such as autoimmune diseases (e.g., rheumatoid arthritis, type 1 diabetes, multiple sclerosis, celiac disease), inflammatory bowel diseases (IBD) (e.g., Crohn’s disease, ulcerative colitis), metabolic disorders (e.g., obesity, insulin resistance, type 2 diabetes), neurological conditions (e.g., Alzheimer’s, autism, depression) and food sensitivities or allergies.

2. Intestinal Fatty Acid-Binding Protein (I-FABP)

I-FABP is a small cytoplasmic protein found in intestinal epithelial cells. It plays a role in fat absorption and metabolism, but more importantly, it serves as a biomarker for intestinal epithelial damage. When intestinal cells are injured due to inflammation, ischemia, infections, or increased gut permeability, I-FABP is released into the bloodstream, making it a useful indicator of gut barrier integrity and epithelial cell damage.

I-FABP can be detected in blood serum and urine. Higher levels suggest acute or chronic intestinal injury. Elevated protein levels can be associated with:

• Inflammatory bowel diseases (IBD) – Crohn’s disease, ulcerative colitis
• Celiac disease – Damage due to gluten exposure
• Ischemic bowel disease – Reduced blood flow to the intestines
• Infections – Bacterial, viral, or parasitic gut infections
• Sepsis and systemic inflammation – Gut-derived endotoxemia
• Metabolic and autoimmune diseases – Type 2 diabetes, rheumatoid arthritis, and other chronic conditions linked to gut permeability

3. Lactulose/Mannitol Test

The Lactulose/Mannitol Test (L/M Test) is a non-invasive and widely used method to assess intestinal permeability. It helps to determine the ability of the small intestine to selectively absorb nutrients while preventing harmful substances from entering the bloodstream.

The test involves drinking a solution containing two types of sugar:

• Mannitol – A small sugar molecule that is easily absorbed by healthy intestinal cells, reflecting normal nutrient absorption.
• Lactulose – A larger sugar molecule that is poorly absorbed and should remain in the gut unless the intestinal barrier is compromised.

After six hours, a urine sample is collected to measure the amount of each sugar excreted. In a healthy intestine, only a small amount of lactulose is absorbed into the bloodstream. When the gut permeability is increased, larger amounts of lactulose enter the bloodstream through the intestinal mucosa. The ratio of lactulose to mannitol in the urine can give an indication of the severity of the leaky gut. A high ratio of lactulose to mannitol indicates increased intestinal permeability.

4. Claudins & Occludin

Claudins and occludin are essential tight junction proteins that regulate intestinal permeability, ensuring that the gut barrier selectively allows nutrients to pass while preventing harmful substances, such as toxins and bacteria, from entering the bloodstream. These proteins play a crucial role in maintaining gut homeostasis and preventing leaky gut syndrome, which is implicated in various inflammatory and autoimmune diseases.

Tight junctions are protein complexes that form a seal between intestinal epithelial cells. They help maintain the structural integrity of the gut barrier, controlling the movement of molecules between cells (paracellular transport). Claudins are a large family of proteins that determine the permeability of tight junctions, either strengthening the barrier (e.g., Claudin-1, Claudin-4) or loosening it (e.g., Claudin-2). Occludin is a regulatory protein that reinforces tight junction stability and helps control immune signaling and epithelial repair. Together, claudins and occludin ensure a balanced barrier function, preventing intestinal hyperpermeability while allowing proper nutrient absorption.

Changes in claudins and occludin can be assessed through (i) gene expression analysis (e.g., RT-PCR, microarrays), (ii) protein detection methods (Western blot, ELISA, immunofluorescence), or (iii) biopsy of samples.

Altered claudin and occludin levels have been shown to be associated with:

• Leaky Gut Syndrome – Increased permeability due to reduced occludin and altered claudin expression, allowing toxins (e.g., lipopolysaccharides, LPS) to enter the bloodstream.
• Inflammatory Bowel Diseases (IBD) – Crohn’s disease & ulcerative colitis show disrupted tight junction proteins, contributing to chronic inflammation.
• Autoimmune Diseases – Conditions like rheumatoid arthritis (RA), multiple sclerosis (MS), and type 1 diabetes are linked to intestinal barrier dysfunction.
• Metabolic Disorders – Obesity, insulin resistance, and type 2 diabetes have been associated with tight junction protein dysregulation.
• Neuroinflammation & Brain Disorders – Gut permeability and altered tight junction proteins may influence conditions like Alzheimer’s disease, autism, and depression (gut-brain axis connection).

5. Faecal Calprotectin

Faecal calprotectin is a non-invasive biomarker primarily used to assess intestinal inflammation, but it also plays a key role in evaluating gut barrier integrity. As a calcium- and zinc-binding protein, calprotectin is released by activated neutrophils (specific white blood cells) in response to gut inflammation and epithelial damage, making it a valuable marker for intestinal permeability dysfunction.

Zonulin vs. I-FABP vs. Lactulose/Mannitol Test vs. Claudins & Occludin  vs. faecal calprotectin: Key Differences

Assessing gut permeability and barrier integrity is crucial for understanding its role in systemic inflammation, autoimmune diseases, and metabolic disorders. Several biomarkers help evaluate gut health, each with distinct mechanisms and applications. Zonulin regulates tight junctions between intestinal cells, making it a useful but indirect marker of gut permeability. I-FABP is released when intestinal epithelial cells are damaged, providing a direct measure of acute gut injury. The Lactulose/Mannitol Test is a functional assessment of a leaky gut, measuring how well sugars pass through the gut lining via urine analysis. Claudins & occludin, key tight junction proteins, help assess structural gut integrity at the molecular level, often through gene expression analysis. Faecal calprotectin, released by activated neutrophils, is a marker of gut inflammation, but is often used as an additional marker for gut permeability.

Each of these biomarkers provides unique insights, and together, they offer a comprehensive picture of gut health and its connection to systemic diseases.

 

Biomarkers of Gut Epithelia Integrity in ENDOTARGET

The ENDOTARGET project explores how gut permeability contributes to systemic inflammation and rheumatic diseases like osteoarthritis (OA), rheumatoid arthritis (RA), and spondyloarthritis (SpA). By analysing a combination of these biomarkers, researchers aim to (i) identify gut barrier dysfunction in patients with rheumatic diseases, (ii) understand how intestinal inflammation and endotoxemia impact systemic immune activation, and (iii) develop gut-targeted interventions to reduce chronic inflammation and improve joint health.

 

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Matei DE, et al. Intestinal barrier dysfunction plays an integral role in arthritis pathology and can be targeted to ameliorate disease. Med. 2021. doi: 10.1016/j.medj.2021.04.013

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