This study investigates the bioavailability of iron in food using a two-chamber cell culture system with enterocytes. The process involves simulating gastric and intestinal digestion to assess how iron is absorbed by specialized cells in the small intestine.
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.
Weigh out the sample in a sterile 50-milliliter centrifuge tube. Adjust the pH using hydrochloric acid, and add 10 milliliters of physiological saline containing 140 millimolar sodium chloride and 5 millimolar potassium chloride. Then, set the gastric digestion process by adding 0.5 milliliters of the prepared porcine pepsin solution to the sample.
Next, incubate on a rocking shaker at a gentle setting, for 1 hour at 37 degrees Celsius, and initiate the intestinal digestion process of each sample by adjusting the pH to 5.5-6.0 with 1.0 molar sodium bicarbonate.
Add 2.5 milliliters of the pancreatic-bile solution to each sample tube, and adjust the pH to 6.9-7.0 with 1.0 molar sodium bicarbonate.
Using the solution containing 140 millimolar sodium chloride and 5 millimolar potassium chloride, add liquid so that each tube contains exactly 15 grams of total material.
Now, transfer 1.5 milliliters of each intestinal digest into the upper chamber of the well containing the Caco-2 cells of the 6-well culture plate. Replace the plate cover, and incubate at 37 degrees Celsius in 5% carbon dioxide on a rocking shaker at 6 oscillations per minute, for 2 hours.
Remove the insert ring with the digest. Then, add 1 milliliter of minimum essential medium at pH 7 to each well, and keep the plate back in the incubator for 22 hours.
Now, remove the cell culture medium and add 2 milliliters of 18 mega ohm (MΩ) water to the cell monolayer. Transfer it to a sonicator and harvest the entire cell lysate into microcentrifuge tubes for cell protein and cell ferritin analyses.
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