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United States Department of Agriculture

Agricultural Research Service

Title: Bathophenanthrolene Disulfonic Acid and Sodium Dithionite Effectively Removes Surface Bound Iron from Caco-2 Cell Monolayers

Authors
item Glahn, Raymond
item Gangloff M B, - CORNELL UNIVERSITY
item Van Campen, Darrell
item Miller D D, - CORNELL UNIVERSITY
item Norvell, Wendell

Submitted to: Journal of Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 19, 1995
Publication Date: N/A

Interpretive Summary: Iron deficiency is generally considered to be the most prevalent nutritional deficiency in the U.S. and in the world. The high prevalence of iron deficiency is due, in part, to poor bio-availability (utilization) of dietary iron. Efforts to improve bioavailability are hampered by a lack of methods for assessing bioavailability that are rapid, inexpensive and relevant. We are attempting to develop such a method by utilizing cultures of a cell line known as Caco-2 cells. These cells, when grown in culture, resemble the absorptive cells of the small intestine, both anatomically and physiologically. However, in earlier studies, we found that certain forms of iron apparently precipated on the cell surface and this iron was not removed by normal washing and rinsing techniques. In order to utilize these cell cultures for estimating bioavailability, it is essential to remove this surface-bound iron. In this paper, we report on a method that we developed which removes surface-bound iron without any apparent damage to the cell membranes. This procedure allows us to continue with the development of Caco-2 cell cultures as a means of estimating iron bioavailability and could, ultimately, help to improve bioavailability of iron in plant food and to reduce the incidence of iron deficiency.

Technical Abstract: Iron uptake by Caco-2 cell monolayers is commonly assessed by incubating the cells under radiolabeled iron solutions, removing the radiolabeled solution, rinsing to stop uptake, and quantitating the radioactivity retained by the cells. It is therefore essential to differentiate between iron that is non-specifically bound to the cell surface from that which has been absorbed by the cell. We report here on a method for removal of surface-bound iron from Caco-2 monolayers. We utilize a 140 mmol/L NaCl, 10 mmol/L PIPES, pH 6.7 solution containing 5.0 mmol/L sodium dithionite (Na2S2O4) and 5.0 mmol/L bathophenanthroline disulfonic acid (BPDS) to reduce, remove and chelate iron bound to the cell surface. We validate our method by demonstrating the removal of 97% of an insoluble iron complex from the apical surface of Caco-2 cell monolayers. Our data indicate that the removal solution does not damage the apical membrane and thereby does not have access to intracellular iron; thus, only surface bound iron is removed. The remaining cell-associated iron represents that which has been absorbed by the cell. We demonstrate the absorption and non-specific binding of iron from Fe complexes of both ferrous and ferric forms, and show that the use of iron removal treatment resulted in uptake measurements which agree more closely with accepted principles of iron uptake by intestinal epithelium. The use of the iron removal method reported in this paper should provide investigators with a valuable tool for accurately determining iron uptake by epithelial cells in culture.

Last Modified: 8/22/2014