Automated assay for non-transferrin-bound iron in serum samples.

Non-transferrin-bound iron (NTBI) is a powerful promoter of free radical damage and highly toxic to biological systems, resulting in oxidative damage to proteins, lipids and DNA. This assay is based on the binding of serum NTBI by the chelator nitrilotriacetic acid (NTA) and measurement of the ultrafiltrated Fe-NTA complex with the ferrozine reagent kit by a biochemical analyzer. To determine NTBI at extremely low concentrations, the program parameters for serum iron measurement were modified. Linearity was up to 15 μmol/L with analytical recovery of 93%-103%. The limit of detection was 0.076 μmol/L. The within-run coefficient of variation was 2.37%, 1.23%, and 0.812% at concentrations of 0.338, 1.717, and 5.916 μmol/L, respectively. NTBI concentrations measured after exercise in samples obtained from 14 rowers, divided into two groups, were substantially higher in all samples. The median NTBI concentrations (range) before and after exercise were 0.197 (-0.11 to 0.58), and 3.353 (2.39-8.97) μmol/L, respectively, in older rowers and 0.197 (-0.18 to 1.17), and 1.360 (0.47-2.49) μmol/L, respectively, in younger rowers. With the described modification for serum iron determination, NTBI can be measured with high sensitivity and specificity. The data presented are illustrative examples of the applicability of this assay.