Measurement of plasma hydroperoxide concentrations by the ferrous oxidation-xylenol orange assay in conjunction with triphenylphosphine.

We describe the application of the FOX2 (ferrous oxidation in xylenol orange, version 2) method to the measurement of hydroperoxides in plasma. Authentic plasma hydroperoxides can be determined by a strategy in which the hydroperoxide reductant, triphenylphosphine, is used to discriminate between the background signal generated by ferric ions present in plasma and that generated by hydroperoxide in plasma. The approach was validated by extraction of total lipids from plasma using ethyl acetate prior to assay with the FOX2 reagent. Plasma from 23 normal individuals contained hydroperoxide in the range of 0.22 to 7.8 microM with a mean of 3.02 microM and a population standard deviation of 1.85 microM. After partitioning with ethyl acetate, plasma hydroperoxide levels ranged from 0.22 to 6.22 microM, with a mean value of 2.52 microM and a population standard deviation of 1.65 microM.