Yamanishi H, Kimura S, Iyama S, Yamaguchi Y, Yanagihara T
Central Laboratory for Clinical Investigation, Osaka University Hospital, Japan.
Clin Chem. 1997 Dec;43(12):2413-7.
We established a method for fully automated measurement of total iron-binding capacity (TIBC) in serum without separation of the unbound excess iron after saturating serum transferrin. After saturation of serum transferrin with an excess amount of iron (first step), the unbound iron was eliminated by formation of a complex with ferrozine, which was used as a chromogenic reagent (second step). For the TIBC assay, iron dissociated from transferrin by shifting the pH to acidic was reacted with ferrozine, and the increase in the absorbance at 570 nm was measured (third step). Because the iron used as a calibrator, which was added to saturate transferrin, reacted completely with ferrozine in the second step (elimination of unbound iron), the change in the absorbance to generate a calibration factor could not be monitored in the third step. To solve this problem, we used N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA) to complex with the iron added to saturate transferrin in the second step. This made it possible to form an iron-ferrozine complex at acidic pH because iron was dissociated from HEDTA at acidic pH. The within-run CVs of this method were 0.66-2.43% at 17.7-77.0 mumol/L, and the day-to-day CVs were 1.06-1.57% at 29.9-60.4 mumol/L (n = 10). The correlation between the values obtained with this method (y) and those from the direct TIBC assay, which involved removal of unbound iron by ion-exchange resin (x), was: y = 0.963x + 0.29 mumol/L (r = 0.973, Sy/x = 2.83, n = 59), and with the TIBC values calculated from the serum iron concentrations and the unbound iron-binding capacities measured by a direct colorimetric method (x) was: y = 1.01x - 1.06 mumol/L (r = 0.994, Sy/x = 1.66, n = 51).
我们建立了一种在血清转铁蛋白饱和后无需分离未结合过量铁即可全自动测量血清总铁结合力（TIBC）的方法。在用过量铁使血清转铁蛋白饱和后（第一步），通过与用作显色剂的亚铁嗪形成复合物来消除未结合的铁（第二步）。对于TIBC测定，通过将pH调至酸性使从转铁蛋白解离的铁与亚铁嗪反应，并测量570nm处吸光度的增加（第三步）。由于用作校准物的铁在第二步（消除未结合的铁）中与亚铁嗪完全反应，因此在第三步中无法监测吸光度的变化以生成校准因子。为了解决这个问题，我们使用N-(2-羟乙基)乙二胺-N,N',N'-三乙酸（HEDTA）在第二步中与添加的使转铁蛋白饱和的铁形成复合物。这使得在酸性pH下能够形成铁-亚铁嗪复合物，因为铁在酸性pH下从HEDTA解离。该方法的批内变异系数在17.7 - 77.0μmol/L时为0.66 - 2.43%，批间变异系数在29.9 - 60.4μmol/L时为1.06 - 1.57%（n = 10）。用该方法获得的值（y）与通过离子交换树脂去除未结合铁的直接TIBC测定法获得的值（x）之间的相关性为：y = 0.963x + 0.29μmol/L（r = 0.973，Sy/x = 2.83，n = 59），与通过血清铁浓度和直接比色法测量的未结合铁结合能力计算得到的TIBC值（x）之间的相关性为：y = 1.01x - 1.06μmol/L（r = 0.994，Sy/x = 1.66，n = 51）。
