Iron–ascorbate complex formation and redox behavior in Tris buffer under aerobic and anaerobic conditions: relevance to iron-deficiency anemia

Abstract

Iron–ascorbate (Fe–Asc) is a clinically approved drug for iron-deficiency anemia (IDA), yet its formation and redox behavior under varying conditions remain poorly understood. Herein, we report the first comprehensive examination of Fe–Asc formation and redox reactivity in Tris buffer as a function of stoichiometry, pH, and oxygen availability. Oxidation of Fe(II) in air-saturated buffer accelerates with pH, yielding multiple species, primarily di- and tri-nuclear clusters. Notably, FeSO₄ with AscH produces a distinct purple complex (λ_max = 510 nm), whose assembly depends critically on O₂, 1:3 stoichiometry of Fe: AscH, and buffer pH 7.5, whereas no such species forms anaerobically. ESI-MS suggests trinuclear clusters, whose stability and redox dynamics are monitored by time-resolved absorbance. The purple color disappears with dithionite or H₂O₂, but dithionite-treated samples regain color upon air exposure, demonstrating redox reversibility. Electrochemically, FeSO₄ shows cathodic (-0.32 V) and anodic (-0.06 V) peaks vs. Ag/AgCl, but the cathodic current decreases markedly in presence of AscH, reflecting Fe–Asc interactions. EPR spectra of FeSO₄ and FeSO₄/AscH are similar, with g = 9.43, 4.37 (high-spin Fe(III), S = 5/2) and g = 1.92 (S = ½, [Fe(III)–Fe(II)] coupling). These results suggest the formation of mixed valence trinuclear clusters with a dynamic ligand environment.

Graphical Abstract

The interaction between Fe and AscH with a specific stoichiometric ratio (Fe: AscH; 1:3), in Tris-buffer at pH7.5 under aerobic conditions, forms a purple complex, suggested as a mixed valence cluster, whereas anaerobic or dithionite-treated purple solutions yield a colorless solution that can reversibly interconvert with the purple form.