Iron is an essential metal in the body, but its overload causes various organ damages through free radical productions. When the balance of iron metabolism is collapsesed by a prolonged transfusion and iron medication etc, transferrin (Tf) will be saturated and non-Tf bound iron (NTBI) will appear in the serum. It has been reported that NTBI relates to oxidant stress and cellular damages. Concerning the measurement of NTBI in blood, there are several problems including the expensive consumables and the long time to measure. Moreover, the method of measuring NTBI was extremely complicated, so that very limited institutions can use this at the present. Among several methods, we focused on high-performance liquid chromatography (HPLC)-based NTBI assay. The principle of measurement of HPLC method is bases on Gosriwatana I et al. (Anal Biochem, 1999); NTBI in serum was initially chelated by NTA and iron was transferred to another chelator, CP22, which had chromogenic effect when bound to iron. At last, the level of iron was quantitated by HPLC system. We improved the sensitivity of this method and reported by extreme reducing background iron contamination (Sasaki K et al. Mol Med Report, 2011). However, HPLC method still needs some improvement, because this method requires the samples and takes long time to measure, so that the many sample handling is difficult. Therefore, the novel measuring system within automated analyzer equipped in many hospitals is desired. From such a background, we aimed to develop the novel NTBI measuring reagent corresponding to the automatic analyzer by modifying the HPLC method.
We developed the novel NTBI measuring system using NTA and Nitroso-xxxx (PSAP) as the chromogen instead of CP22. This system does not need any pre-treatment of serum samples. Then, we examined the basic performance of the novel NTBI reagent, and compared to HPLC method. Data analyses were performed using HITACHI 7700 auto analyzer. The following points were examined: (1) reproducibility (N=20), (2) comparison between the linearity of actual measured data and that of theoretically calculated data, (3) detection limit, (4) relation between the Tf saturations and NTBI values, (5) comparison of the value measured by the HPLC method and the value measured using novel NTBI measuring system.
Reproducibility was satisfactory (NTBI levels of pooled serum were 0.491±0.190μM). And measured values using this novel system showed a dose-dependent curve under the iron concentrations between 0 to 5μM. The detection limit was found to be 0.5μM using ±2.6SD at zero concentrations and was also able to detect the NTBI in blood in healthy person serum. In serum, when Tf saturation was approaching to 100%, NTBI turns into high values. When NTBI values of the serum obtained from the patients with written informed consent were measured, positive correlation (R2=0.935) was observed between the Tf saturation and NTBI. Moreover, the data obtained by HPLC method correlate positively with those determined by novel measuring system (R2=0.876). These should indicate that our novel measuring system for NTBI possess comparable efficiency compared with the HPLC method. Moreover, our new method should have quite attractive advantages: (A) that can measured approximately 800 samples in 1 hour, (B) that does not need any pre-treatment of sample serum, and (C) that does not need any expensive consumables, meaning excellent cost-benefit. Therefore our new method for the NTBI measuring using automatic analyzer must be useful and powerful for the research to elucidate the clinical importance of NTBI in various diseases in the near future.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.