IRON SPECIATION AT PHYSIOLOGICAL PH IN MEDIA CONTAINING ASCORBATE ANDOXYGEN

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 speci es present under the differing conditions were investigated by electro n paramagnetic resonance (EPR) and Mossbauer spectroscopy and by gel-f iltration chromatography. At physiological pH (6-8-7.4) iron ascorbate solutions rapidly form mononuclear chelatable Fe2+ species as reflect ed by indicator studies and EPR. Mossbauer spectroscopy fails to detec t any Fe2+ species. EPR studies show a time-dependent decrease in rhom bic Fe3+, particularly in oxygenated buffers, consistent with a conver sion 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 indicat e the inherent instability of Fe-ascorbate solutions above neutral pH and clearly have important implications in the use of ascorbate in stu dies of Fe physiology.