Characterizing ferrous versus ferric iron in Alzheimer's disease using X-ray fluorescence imaging and XANES spectroscopy

Background The accumulation of iron, such as ferrous Fe 2+ , in the Alzheimer's disease (AD) brain may contribute to neurodegeneration by driving oxidative stress. While elevated iron in AD has been shown, the oxidation state of iron and its regional distribution in AD, particularly in the hippocampus, is unclear. Objective To characterize the oxidation state and spatial distribution of iron in the hippocampus of AD and control brains, and to assess the effect of tissue thawing on ferrous iron measurements. Methods We utilized X-ray fluorescence imaging and X-ray absorption near edge structure spectroscopy to localize and analyze iron deposition in fresh-frozen human hippocampal specimens stratified by AD disease stage. To assess the effect of thawing on iron oxidation, we used a cryo-chamber to keep three specimens frozen while their respective deposits were being scanned. These specimens were then allowed to thaw and their same deposits were rescanned for comparison. Results Compared to control brains, AD specimens exhibited elevated levels of ferrous iron (Fe 2 + ) in the cornu ammonis 1 (CA1)-subiculum subfields—regions known to degenerate early in AD. We also measured a decrease in Fe 2+ levels in AD and control specimens scanned after being thawed. Conclusions Our findings support the association between elevated Fe 2+ and AD, consistent with existing hypotheses linking redox-active iron to oxidative stress and neuroinflammation. The observed reduction in Fe 2+ levels following thawing suggests that studies using thawed brain samples may underestimate Fe 2+ levels.