Non-transferrin-bound iron species in the serum of hypotransferrinaemic mice

Serum from homozygous hypotransferrinaemic mice (a mixed group of males and females, aged 6-8 wk) was found to contain low levels of iron (mean 0.9±0.5 μM (SEM, n=4), as assayed by conventional serum iron assays. Similarly, low levels of non-transferrin-bound iron were determined with a nitrilotriacetate chelation assay (1.3±0.4 μM, n=4) (Singh, S., Hider, R.C. and Porter, J.B. (1990) Analytical Biochemistry 186, 320-323). Mononuclear Fe (citrate) was undetectable by electron paramagnetic resonances spectroscopy (EPR). Significantly larger quantities of iron (16±5 μM, n=8) were detected by the bleomycin assay (Gutteridge, J.M.C., Rowley, D.A. and Haliwell, B. (1981) Biochemical 199, 263-265), while non-haem iron assay or atomic absorption spectrophotometry revealed up to 96 μM iron. Haemoglobin iron was detected at approximately 10 μM by spectrophotometry. Ferr-haem was undetectable by EPR spectroscopy. Serum ferritin levels of 641±128 μg/l (n=14) in hypotransferrinaemic mice (wild-types 44±6 μg/l, n=14) were observed and these cannot account for the non-transferrin-bound iron. Hypotransferrianemic mouse serum therefore contains large quantities of non-transferrin-bound iron which is unreactive in some assays used to detect such iron in human iron overload. Fractionation by Sephadex G200 chromatography revealed three distinct species with apparent molecular weights of ≥ 150 kDa 40-80 kDa and 1-5 kDa. The iron may be distinguished from known extracellular iron proteins and haem-proteins by its availability to hot acid extractions.