A new automated colorimetric method for measuring total oxidant status

Objectives: To develop a new, colorimetric and automated method for measuring total oxidation status (TOS). Design and methods: The assay is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in acidic medium and the measurement of the ferric ion by xylenol orange. The oxidation reaction of the assay was enhanced and precipitation of proteins was prevented. In addition, autoxidation of ferrous ion present in the reagent was prevented during storage. The method was applied to an automated analyzer, which was calibrated with hydrogen peroxide and the analytical performance characteristics of the assay were determined. Results: There were important correlations with hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide solutions (r = 0.99, P < 0.001 for all). In addition, the new assay presented a typical sigmoidal reaction pattern in copper-induced lipoprotein autoxidation. The novel assay is linear up to 200 μmol H2O2 Equiv./L and its precision value is lower than 3%. The lower detection limit is 1.13 μmol H2O2 Equiv./L. The reagents are stable for at least 6 months on the automated analyzer. Serum TOS level was significantly higher in patients with osteoarthritis (21.23 ± 3.11 μmol H2O2 Equiv./L) than in healthy subjects (14.19 ± 3.16 μmol H2O2 Equiv./L, P < 0.001) and the results showed a significant negative correlation with total antioxidant capacity (TAC) (r = −0.66 P < 0.01). Conclusions: This easy, stable, reliable, sensitive, inexpensive and fully automated method that is described can be used to measure total oxidant status.