Iron speciation at physiological pH in media containing ascorbate and oxygen

Abstract

The stability of iron ascorbate solutions was investigated, under both anaerobic and aerobic conditions, with the Fe²⁺ and Fe³⁺ indicators, respectively ferrozine and mimosine, at different pH values. The species present under the differing conditions were investigated by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy and by gel-filtration chromatography. At physiological pH (6·8–7·4) iron ascorbate solutions rapidly form mononuclear chelatable Fe³⁺ species as reflected by indicator studies and EPR. Mössbauer spectroscopy fails to detect any Fe²⁺ species. EPR studies show a time-dependent decrease in rhombic Fe³⁺, particularly in oxygenated buffers, consistent with a conversion to polynuclear Fe. O₂ uptake studies show that the conversion of Fe²⁺ to Fe³⁺ in Fe–ascorbate solutions at pH > 7·0 was accompanied by rapid O₂ consumption but preceded depletion of ascorbate. Addition of high concentrations of mannitol (50–200 mM) reduces the O₂ consumption and partly stabilizes the rapidly chelatable Fe form. Gel filtration studies show that the oxidation of Fe–ascorbate solutions at pH 7·4 is accompanied by an increase in the apparent relative molecular mass of the Fe, presumably due to Fe polymer formation. These studies indicate the inherent instability of Fe–ascorbate solutions above neutral pH and clearly have important implications in the use of ascorbate in studies of Fe physiology.