The Fluorescein Isothiocyanate-Dextran Permeability Assay measures the integrity of various protective biological barriers, such as the intestinal lining and the blood-brain barrier. These complex structures regulate the passage of substances, ensuring nutrients enter while toxins and pathogens are kept out. A loss of integrity is often associated with the onset or progression of numerous diseases. The assay provides a quantitative measure of barrier function by introducing a tracer molecule and monitoring its movement across the membrane.
The Role of Fluorescein Isothiocyanate and Dextran
The assay depends on two components that create a measurable tracer molecule. Dextran is a high-molecular-weight, inert polysaccharide that is not absorbed or metabolized by the body. Researchers select Dextran because its size prevents it from naturally crossing an intact biological barrier, making it an ideal marker for leakage. Dextran is available in various molecular weights (e.g., 4 kDa or 10 kDa), which allows researchers to assess the size-selectivity of the barrier’s pores. The second component, Fluorescein Isothiocyanate (FITC), is a small, fluorescent dye chemically attached to the Dextran molecule. This attachment makes the Dextran easily detectable, as the FITC tag emits light at a specific wavelength when excited by a light source.
Measuring Barrier Integrity: The Mechanism
The fundamental principle of the assay is to introduce the tracer on one side of a barrier and measure how much appears on the other side. When studying intestinal permeability, the FITC-Dextran tracer is typically administered orally, often via oral gavage in animal models. The tracer travels through the gastrointestinal tract, encountering the intestinal lining.
In a healthy system, tight junctions prevent the large Dextran molecule from passing into the bloodstream. If the barrier is compromised, Dextran leaks between the cells, migrating from the gut lumen into the circulatory system. After a set period (typically one to four hours), a blood sample is collected and processed into plasma. The plasma is analyzed using a fluorometer or spectrophotometer, which measures the intensity of the green fluorescence emitted by the FITC tag. A high fluorescence reading in the plasma correlates to a significant amount of Dextran having crossed the barrier, providing a quantitative measure of leakiness.
Primary Applications in Permeability Studies
The FITC-Dextran assay investigates the function of various biological interfaces. One primary application is the study of intestinal permeability, often called “leaky gut.” Researchers use this assay to study digestive disorders like Inflammatory Bowel Disease (IBD) and Celiac disease, and the effects of diet, probiotics, or pharmaceuticals on the gut lining.
The assay also assesses the integrity of the Blood-Brain Barrier (BBB), a highly selective membrane protecting the central nervous system. In neurological research, the passage of FITC-Dextran into the brain tissue or cerebrospinal fluid indicates a breakdown of the BBB, relevant to diseases like stroke, multiple sclerosis, and Alzheimer’s disease.
Beyond internal barriers, the technique is valuable in drug testing and toxicology studies to evaluate the effects of new compounds. It can be adapted to in vitro models, such as cell monolayers, to assess whether a chemical compound damages the epithelial or endothelial cells. This application helps quantify a compound’s potential toxicity or its ability to alter cellular barrier function.
Interpreting Permeability Measurements
The final step involves translating the raw fluorescence data. The fluorometer provides a measurement in relative fluorescence units (RFU) or is converted into a concentration, typically expressed in micrograms of FITC-Dextran per milliliter (\(\mu\)g/mL) of the sample. A high concentration of the tracer in the blood or urine indicates high barrier permeability, signifying a compromised barrier.
Conversely, a low concentration suggests the barrier is functioning effectively and restricting the passage of Dextran. To ensure accuracy, researchers establish a standard curve using known concentrations of FITC-Dextran to precisely quantify the amount in the test samples. Results are reported as a comparison between different groups, such as a treated group versus a control group, allowing for an objective determination of whether a factor has caused a significant change in barrier function.