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Updated: Jun 16, 2026

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability
Published on: April 28, 2022
Bioavailability of Ferric Pyrophosphate and Ferric Orthophosphate with/without Extrusion and/or Citric Acid and/or
Jiejia Zhang1, David B Akinbo1, Erin J Ward1
1School of Health Sciences, College of Human Health and Sciences, Kansas State University, Manhattan, KS, United States.
Background:
Iron absorption of fortified rice and/or extruded rice flour could be significantly increased with the addition of ferric pyrophosphate (FePP)/micronized FePP (μFePP)/ferric orthophosphate (FePO4), citric acid (CA), and trisodium citrate (TSC) at low (1:0.1:2.1) and high (1:0.3:5.5) molar ratios. Thus, 2 rat studies and a Caco-2 cell study were carried out to examine the iron bioavailability of different formulations and food processing strategies.
Objectives:
To determine the iron bioavailability of rice and extruded rice flour fortified with the different formulations of FePP/μFePP/FePO4, CA, and TSC in rats and the INFOGEST digestion/Caco-2 cell model.
Methods:
In rat studies, Sprague-Dawley rats were randomly assigned iron-fortified rice diets using the prophylactic-preventative method. The iron bioavailability was determined by measuring the concentrations of hematological parameters and hepatic iron. In the cell-culture study, iron-fortified extruded rice flour was digested with the INFOGEST digestion method. The iron solubility was analyzed with inductively coupled plasma-optical emission spectrometry. The iron bioavailability was determined by measuring ferritin concentrations.
Results:
In rat study 1, hemoglobin and hepatic iron levels were significantly higher in the FePP and μFePP groups compared with the FePO4 and control groups. In rat study 2, μFePP and high ratio CA and TSC FePP groups significantly increased hepatic iron concentrations compared with the high ratio CA and TSC FePO4 and control groups. In the cell-culture study, among coextruded samples, the ferritin concentrations of high ratio samples were significantly higher than those of low and zero ratio samples. Among mixed samples, the high ratio FePO4 had significantly higher ferritin concentration than the zero ratio FePO4.
Conclusions:
The iron bioavailability of FePP and FePO4 was improved with CA and TSC at a high molar ratio. High ratio FePP and μFePP were superior to high ratio FePO4 in increasing hepatic iron concentrations.
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