Evidence for an Enhancing Effect of Alginate on Iron Availability in Caco‑2 Cells Anna A. Wawer, † Paul A. Sharp, § Natalia Perez-Moral, # and Susan J. Fairweather-Tait* ,† † Department of Nutrition, Norwich Medical School, University of East Anglia, Norwich NR4 7TJ, United Kingdom § School of Biomedical and Health Sciences, Nutritional Sciences Division, King’s College, London SE1 9NH, United Kingdom # Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom ABSTRACT: The potential use of alginates as a vehicle for water-soluble (bioavailable) iron for fortifying food products was examined using a Caco-2 cell model system. Cell monolayers were exposed to alginates with various mannuronic to guluronic acid ratios at three different concentrations, and cellular ferritin was measured as a surrogate marker of iron uptake into the cell. Ferritin concentrations were significantly higher when the cells were treated with ferric ammonium citrate and 0.5 and 1% w/v (but not 0.1%) alginate, but were unaffected by mannuronic/guluronic acid ratios. The enhancing effect of ascorbic acid was maintained with 0.1% alginate and significantly increased with 0.5 and 1% alginate, whereas the inhibitory effect of tannic acid was significantly reduced with 0.5% alginate. Alginate beads delivered available iron to Caco-2 cells, indicating that they are a promising vehicle for soluble iron with potential use in food fortification programs. KEYWORDS: iron bioavailability, alginate, Caco-2 cells, ferritin, ascorbic acid, tannic acid ■ INTRODUCTION Estimates made by the World Health Organization suggest that anemia affects 1.62 billion people, that is, one-fourth of the world’s population, with preschool children and pregnant women being at greatest risk. 1 Because iron deficiency is a common cause of anemia, fortification of foods with iron is considered to be a sustainable and realistic way to reduce the risk of iron-deficiency anemia 2 but is a challenge for the food industry. This is because water-soluble forms of iron, which are generally more bioavailable than nonsoluble iron compounds, 3 often cause adverse organoleptic changes when added to foods. 4 A potential strategy for overcoming this problem is the use of water-soluble iron compounds that are protected by a water-resistant barrier. Alginates, natural biopolymers present in brown seaweed, have been shown to bind divalent and trivalent cations 5,6 and can form a stable complex with ferric iron over a range of different pH values. 6 They may therefore be a useful vehicle when water-soluble iron compounds are employed to fortify food products. A model system commonly used for an iron bioavailability screening employs Caco-2 cells. 7-9 Once differentiated, these cells share many characteristics of normal absorptive enter- ocytes. 10,11 Furthermore, if a food/compound is subjected to simulated gastric and small intestinal digestion, with the associated changes in pH, 12 before application to the Caco-2 model system, 8 the similarity to the in vivo situation and its predictive value improve. 13,14 This system is a valuable tool for screening different iron sources to rank them for potential bioavailability and for investigating mechanisms of absorption in humans. 7,15 Alginates are composed of various ratios of two different acids, D-mannuronic and L-guluronic acid (M:G), and due to the variable length of the polymer chains, they exhibit wide physicochemical properties 16 and are used in a wide variety of applications, including drug delivery systems, 17 antireflux preparations, 18 and thickening agents in food products. 19 The effect of alginates, used as thickening agents in infant formulas, on iron, zinc, and calcium availability has been investigated in vitro; the bioavailability of iron and zinc increased, whereas that of calcium decreased in the presence of alginates. 20 In comparison, results from a study carried out in six ileostomy subjects showed that alginates, when administered in frozen milkshake and jam, had no significant effect on iron absorption, but the study was underpowered. 21 The aim of the present study was to evaluate the effects of food grade sodium alginates (E401) on iron uptake into Caco-2 cells. We designed experiments to determine if they could be used as a delivery system (protective barrier) for soluble iron. Specifically, Caco-2 cell studies were carried out to evaluate the effects of various ratios of mannuronic and guluronic acids on iron uptake from ferric ammonium citrate (FAC) and ferrous gluconate (FeG) in the presence of an iron enhancer, ascorbic acid (AA), and an iron inhibitor, tannic acid (TA). ■ MATERIALS AND METHODS Caco-2 Cell Culture Procedures. Unless otherwise stated, all reagents were purchased from Sigma-Aldrich, UK. Caco-2 cells (HTB- 37) were obtained from American Type Culture Collection (Manassas, VA, USA) at passage 20 and stored in liquid nitrogen. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen UK; supplemented with 10% fetal bovine serum, 25 mM HEPES solution, 4 mM L-glutamine, and 5 mL 5000 u/mL penicillin/ streptomycillin solution) and were maintained at 37 °C in an incubator with humidified atmosphere consisting of 5% carbon dioxide and 95% air. Received: July 18, 2012 Revised: October 22, 2012 Accepted: October 26, 2012 Published: October 26, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 11318 dx.doi.org/10.1021/jf3031309 | J. Agric. Food Chem. 2012, 60, 11318-11322