Quantification of ferritin-bound iron in murine samples for Alzheimer’s disease studies using species-specific isotope dilution mass spectrometry

Abstract

We have investigated species-specific isotope dilution mass spectrometry (IDMS) for the quantification of ferritin-bound iron in murine serum and brain. Therefore, fresh samples were analyzed using size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Isotopically labeled (57Fe)ferritin was used as calibrant for the quantification of ferritin-bound iron in murine samples. Assessment of the iron load of serum ferritin was impaired by concomitant iron-containing proteins of similar size and shape, which could not be separated by SEC nor centrifugal ultra-filtration. In contrast, ferritin was the main iron-containing protein in cytosolic extracts of murine brain, which showed a total ferritin-bound iron content of (1.05 ± 0.12) µg g−1 (n= 10; U, k= 2). The relative expanded uncertainty achieved for a mass fraction of ca. 1 µg g−1 ferritin-bound iron was 11% (Urel, k = 2). The relative expanded uncertainty of the iron mass fraction of the (57Fe)ferritin spike was 5.7% and represented the major contributing factor to the overall uncertainty. Statistical tests suggested no significant difference in ferritin-bound iron content between mouse brain hemispheres. The presented analytical tool provides low limits of quantification (2.2 ng g−1) and uncertainties (11%, Urel, k = 2), thus enables the quantification of ferritin-bound iron in murine brain extracts with high sensitivity and accuracy. Furthermore, this analytical workflow assures comparability of measurement results across research laboratories. This provides the basis for investigation of the iron loading of ferritin in brain tissue of healthy and Alzheimer’s disease mouse models, which may help answering the question if iron regulation is impaired in Alzheimer’s disease.