Non-transferrin-bound iron species in the serum of hypotransferrinaemic mice
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R J Simpson1, C E Cooper, K B Raja
1Department of Clinical Biochemistry, King's College School of Medicine and Dentistry, London, UK.
Abstract:
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 microM (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 microM, n = 4) (Singh, S., Hider, R.C. and Porter, J.B. (1990) Analytical Biochemistry 186, 320-323). Mononuclear Fe (citrate) was undectable by electron paramagnetic resonance spectroscopy (EPR). Significantly larger quantities of iron (16 +/- 5 microM, n = 8) were detected by the bleomycin assay (Gutteridge, J.M.C., Rowley, D.A. and Halliwell, B. (1981) Biochemical Journal 199, 263-265), while non-haem iron assay or atomic absorption spectrophotometry revealed up to 96 microM iron. Haemoglobin iron was detectable at approximately 10 microM by spectrophotometry. Ferri-haem was undetectable by EPR spectroscopy. Serum ferritin levels of 641 +/- 128 micrograms/l (n = 14) in hypotransferrinaemic mice (wild-types 44 +/- 6 micrograms/l, n = 14) were observed and these cannot account for the non-transferrin-bound iron. Hypotransferrinaemic 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 > or = 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.
Insights
Hypotransferrinaemic mouse serum contains significant non-transferrin-bound iron, unreactive in common human iron overload assays. This iron exists in various molecular weight fractions, distinct from known iron-binding proteins.
Area of Science:
- Biochemistry
- Hematology
- Analytical Chemistry
Background:
- Transferrin is the primary iron transport protein in serum.
- Iron overload disorders involve excess iron accumulation.
- Assaying non-transferrin-bound iron (NTBI) is crucial for diagnosing iron overload.
Purpose of the Study:
- To characterize the iron content in serum from hypotransferrinaemic mice.
- To investigate the nature and reactivity of non-transferrin-bound iron in this model.
- To compare iron detection methods in a low-transferrin state.
Main Methods:
- Conventional serum iron assays
- Nitrilotriacetate chelation assay for NTBI
- Electron paramagnetic resonance (EPR) spectroscopy
- Bleomycin assay
- Atomic absorption spectrophotometry
- Sephadex G200 chromatography
Main Results:
- Hypotransferrinaemic mouse serum showed low transferrin-bound iron but high NTBI levels.
- NTBI reactivity varied significantly across different assays.
- Iron was found in three distinct molecular weight fractions after chromatography.
- Ferritin levels were elevated but insufficient to account for the observed NTBI.
Conclusions:
- Hypotransferrinaemic mouse serum harbors substantial NTBI that is poorly reactive in certain assays.
- This model presents unique challenges for NTBI quantification compared to human iron overload.
- Further characterization of NTBI species is necessary for understanding iron metabolism and overload.
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