Sensitive method for nontransferrin-bound iron quantification by graphite furnace atomic absorption spectrometry

Objective: To establish a sensitive method for measuring nontransferrin-bou nd iron (NTBI) in serum samples using graphite furnace atomic absorption sp ectrometry (GFAAS). Design and methods: Nontransferrin-bound iron (NTBI) was chelated using nit rilotriacetic acid (NTA) and then ultrafiltered according to the method emp loyed by Singh et al. [1]. Serum ultrafiltrates were diluted eightfold with distilled water. NTBI from the Fe-NTA complex present in the serum ultrafi ltrate was measured using GFAAS. Results: Nontransferrin-bound iron (NTBI) and other parameters were measure d in seven patients diagnosed with hereditary hemochromatosis by liver biop sy. Total serum iron, NTBI and transferrin saturation values (ranging from 87% to 90%) were elevated for three of the seven hemochromatosis patients t ested before therapeutic phlebotomy. Six of the seven hemochromatosis patie nts had undergone phlebotomy and revealed normal total serum iron, NTBI and transferrin saturation values. Nine test subjects (not diagnosed with hemo chromatosis) with abnormally high total serum iron and/or ferritin concentr ations exhibited normal NTBI values (less than or equal to0.14 mu mol/L to 0.29 mu mol/L). The detection limit was 0.1 mu mol/L for a 25 muL injection volume. Conclusions: The GFAAS method presented here provides a sensitive assay to quantitate NTBI in serum samples. The method developed is 4 to 5 times more sensitive than the only other GFAAS method [2] and more than an order of m agnitude more sensitive than other colorimetric methods [1,3]. Improvement in sensitivity over the other GFAAS method [2] may be accounted for by diff erences in sample preparation between this method and that of Nielsen et al . [2]. Serum ultrafiltrates in this study were diluted eightfold with disti lled water and mixed with a magnesium nitrate matrix modifier before GFAAS analysis. NTBI results obtained from this study indicate that the plasma ir on pool in hemochromatosis patients awaiting phlebotomy increases to a leve l at which transferrin's ability to bind iron becomes exhausted and elevate d NTBI levels appear in the serum. NTBI can mediate the production of react ive oxygen species and may cause organ damage associated with iron overload . (C) 2001 The Canadian Society of Clinical Chemists. All nights reserved.