Binding of Citrate-Fe(3+) to Plastic Culture Dishes, an Artefact Useful as a Simple Technique to Screen for New Iron Chelators

NaCT mediates citrate uptake in the liver cell line HepG2. When these cells were exposed to iron (Fe(3+)), citrate uptake/binding as monitored by the association of [(14)C]-citrate with cells increased. However, there was no change in NaCT expression and function, indicating that NaCT was not responsible for this Fe(3+)-induced citrate uptake/binding. Interestingly however, the process exhibited substrate selectivity and saturability as if the process was mediated by a transporter. Notwithstanding these features, subsequent studies demonstrated that the iron-induced citrate uptake/binding did not involve citrate entry into cells; instead, the increase was due to the formation of citrate-Fe(3+) chelate that adsorbed to the cell surface. Surprisingly, the same phenomenon was observed in culture wells without HepG2 cells, indicating the adsorption of the citrate-Fe(3+) chelate to the plastic surface of culture wells. We used this interesting phenomenon as a simple screening technique for new iron chelators with the logic that if another iron chelator is present in the assay system, it would compete with citrate for binding to Fe(3+) and prevent the formation and adsorption of citrate-Fe(3+) to the culture well. This technique was validated with the known iron chelators deferiprone and deferoxamine, and with the bacterial siderophore 2,3-dihydroxybenzoic acid and the catechol carbidopa.