Citrate and albumin facilitate transferrin iron loading in the presence of phosphate

Labile plasma iron (LPI) is redox active, exchangeable iron that catalyzes the formation of reactive oxygen species. Serum transferrin binds iron in a non-exchangeable form and delivers iron to cells. In several inflammatory diseases serum LPI increases but the reason LPI forms is unknown. This work evaluates possible pathways leading to LPI and examines potential mediators of apo transferrin iron loading to prevent LPI. Previously phosphate was shown to inhibit iron loading into apo transferrin by competitively binding free Fe³⁺. The reaction of Fe³⁺ with phosphate produced a soluble ferric phosphate complex. In this study we evaluate iron loading into transferrin under physiologically relevant phosphate conditions to evaluate the roles of citrate and albumin in mediating iron delivery into apo transferrin. We report that preformed Fe³⁺-citrate was loaded into apo transferrin and was not inhibited by phosphate. A competition study evaluated reactions when Fe³⁺ was added to a solution with citrate, phosphate and apo transferrin. The results showed citrate marginally improved the delivery of Fe³⁺ to apo transferrin. Studies adding Fe³⁺ to a solution with phosphate, albumin and apo transferrin showed that albumin improved Fe³⁺ loading into apo transferrin. The most efficient Fe³⁺ loading into apo transferrin in a phosphate solution occurred when both citrate and albumin were present at physiological concentrations. Citrate and albumin overcame phosphate inhibition and loaded apo transferrin equal to the control of Fe³⁺ added to apo transferrin. Our results suggest a physiologically important role for albumin and citrate for apo transferrin iron loading.