Quantification of ferritin bound iron in human serum using species-specific isotope dilution mass spectrometry

Abstract

Ferritin is a hollow sphere protein composed of 24 subunits that can store up to 4500 iron atoms in its inner cavity. It is mainly found in the liver and spleen but also in serum at trace levels. Serum ferritin is considered as the best single indicator in assessing body iron stores except liver or bone marrow biopsy. However, it is confounded by other disease conditions. Ferritin bound iron (FBI) and ferritin saturation have been suggested as more robust biomarkers. The current techniques for FBI determination are limited by low antibody specificity, low instrument sensitivity and possible analyte losses during sample preparation. The need for a highly sensitive and reliable method is widely recognized. Here we describe a novel technique to detect serum FBI using species-specific isotope dilution mass spectrometry (SS-IDMS). [⁵⁷Fe]-ferritin was produced by biosynthesis and in vitro labeling with the ⁵⁷Fe spike in the form of [⁵⁷Fe]-citrate after cell lysis and heat treatment. [⁵⁷Fe]-ferritin for sample spiking was further purified by fast liquid protein chromatography. Serum ferritin and added [⁵⁷Fe]-ferritin were separated from other iron species by ultrafiltration followed by isotopic analysis of FBI using negative thermal ionization mass spectrometry. Repeatability of our assay is 8% with an absolute detection limit of 18 ng FBI in the sample. As compared to other speciation techniques, SS-IDMS offers maximum control over sample losses and species conversion during analysis. The described technique may therefore serve as a reference technique for clinical applications of FBI as a new biomarker for assessing body iron status.