A simplified spectrophotometric determination of unsaturated iron binding capacity in serum

Abstract

Transferrin, an iron binding protein, facilitates iron metabolism by transfering iron in serum. Its unsaturated iron binding capacity has been measured by deducting serum iron (SI) from total iron binding capacity (TIBC). For TIBC measurements in the alkaline zone, the procedure has been to add an excess quantity of iron in order to saturate transferrin with iron, and acidify it after removing the surplus iron with a suitable adsorbent. The iron is then released from transferrin in order to make a colorimetric determination. There are also other procedures which are based on direct radio isotope measurements in which transferrin is saturated with a known quantity of ⁵⁹Fe and the surplus is adsorbed in a resin sponge in order to determine the intensity of radioactivity. However, in these methods there were difficulties in that the procedures for removing the surplus iron were complicated, and that special apparatus was necessary for RI. Therefore, ways to make use of transferrin's binding capacity in the alkaline zone have been studied. Transferrin can be saturated with a known quantity of exess iron. The remaining unbound surplus iron can then be fixed quanttaitively so as to obtain the unsaturated iron binding capacity (UIBC) value. As iron ion solution is unstable in the alkaline zone, it is stabilized as nitrilotriacetic acid (NTA) -iron chelates. The remaining surplus iron, viz NTA-iron chelates, is measured by using bathophenanthroline sulfonic acid (BPT).
Iron and BPT chelates have maximum absorbance at a wave length of 535 nm and iron, following Beer's law, up to 600 μg/dl. Under experimental conditions, it has been confirmed that there is a correlation between the binding capacity of transferrin with iron and the stability of iron chelates with NTA, or BPT; (transferrin-BPT) NTA in the range of pH 8.48.7. The error of this method is approximately 15 μg/dl when the concentration of bilirubin is 10 mg/dl and the mean recovery is 94.4%. Results obtained by this method can be correlated well with the results of the adsorbent methods, obtained by calculation from TIBC (light MgCO₃, Amberlite IRA-410 resin adsorption). Thus, UIBC can be measured simply, quickly and accurately: the coefficient of variation is 1.3% with a mean of 190 μg/dl, (obtained by application to human serum measurement). Relatively small amounts of serum samples can be analyzed by this method, and thus it is also suitable for routine clinical determination.