Use of Membrane Cone Filter and Bathophenanthroline for Colorimetry of Free Iron in Human Serum

抄録

To determine free iron in human serum, we first determined total concentrations of iron (I_t μmol/liter) and protein (P_t μg/ml) in the serum precisely with bathophenanthroline and Coomassie Brilliant Blue G-250 (Bio-Rad Protein Assay) by colorimetry. Then, 1.0ml of the serum was centrifuged on a membrane cone filter (Centriflo CF-25) at 1, 050×g for 60min at 4°C, and the serum protein fraction on the membrane cone filter was dissolved in saline and centrifuged again in the same way. The washed serum protein fraction was dissolved in saline, and an aliquot of the solution was subjected to analyses of iron (I_w μmol/liter) and protein (P_w μg/ml). To correct for the free iron adsorbed on the membrane cone filter during the centrifugation and to obtain the free iron concentration (I_f μmol/liter) in the serum, we used the following equation:
I_f(μmol/liter)=I_t-(I_w×P_t/P_w).
The average values of total and free iron in 16 different samples of human fresh sera were determined to be 21.5±6.1μmol/liter and 1.24±0.89 μmol/liter, respectively, by the present standard method; and the values were compared with those of the same sera frozen for 1 week. Free iron in human serum was found to exist as Fe²⁺.