Evaluation of an Automated Spectrophotometric Assay for Reactive Oxygen Metabolites in Serum

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Abstract
The in vivo assessment of free radicals concentration is hampered by their instability and extremely short half-life. The Diacron Reactive Oxygen Metabolites (D-ROM) test is a recently introduced method to evaluate the peroxidation of organic compounds. Since the manual performance of the test provides excessive analytical imprecision, the aim of this study was to evaluate the automation of this test. Within- and between-run imprecision and interference were assessed according to the guidelines proposed by the NCCLS. The reactive oxygen metabolites' (ROM) stability was evaluated in different physical conditions. For within-run and between-run imprecision the coefficients of variation were consistently lower than 5%. The maximum allowable concentration was 28.2 mmol/l, 0.068 mmol/l and 171 mmol/l for triglycerides, haemoglobin and bilirubin, respectively. Serum storage at -20 degrees C provided adequate ROM stability for up to 3 months, whereas storage at 4 degrees C yielded non-reproducible results. In conclusion, our data provide evidence that the D-ROM assay has both an acceptable stability and an adequate imprecision. The automated assay may be regarded as a fast and reproducible method for the quantitative evaluation of oxidative stress. Since it is easily performed, the method is suitable for routine in clinical laboratories and may provide an accurate estimation of oxidative stress in vivo.