Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources

Enterocytes are specialized small intestine cells that play a crucial role in nutrient uptake, including iron absorption.

To assess iron bioavailability - the proportion of ingested iron accessible for physiological functions - take an acidified solution containing the food sample. Now, add pepsin - a protein digestion enzyme, from the stomach. At acidic pH, pepsin initiates gastric digestion by breaking down complex dietary proteins into small peptides.

Add sodium bicarbonate to increase the pH and stop gastric digestion. Add a solution containing pancreatic enzymes and bile to simulate intestinal digestion. Transfer the mixture into the upper portion of a two-chamber cell culture system.

The lower chamber contains enterocytes differentiated from Caco-2 - a colon adenocarcinoma cell line. A dialysis membrane separates the upper chamber from the lower one. The membrane allows the diffusion of low molecular weight species.

The pancreatic enzymes-bile mixture in the upper chamber breaks down carbohydrates, proteins, and lipids into smaller subunits - releasing iron in the form of ferric ions.

Bioavailability enhancers in the food convert the released ferric ions to the ferrous state - increasing their solubility. These ions pass through the membrane to reach the lower chamber.

Metal ion transporters in the cell membrane of enterocytes aid in the uptake of ferrous ions. Once inside, iron binds to apoferritin - an iron storage protein resulting in a complex, ferritin. Harvest the cells to quantify ferritin to assess iron bioavailability.