[The measurement of antioxidant activity in human plasma using cumene hydroperoxide]

We describe a new method using cumene hydroperoxide to determine antioxidant activity (AO) in human plasma. We used a kit (Determiner LPO: Kyowa Medex Co., LTD. Tokyo Japan) for the determination of lipid peroxides in plasma or serum. 30 microliters 1 of sample was mixed with 70 microliters 1 of cumene hydroperoxide (50 nmol/ml) and incubated at 30 degrees C for 120 min before analysis. Samples were mixed with 1.0 ml of reagent-I (Determiner LPO) and incubated at 30 degrees C for 5 min. Then 2.0 ml of reagent-II (Determiner LPO) was added and incubated at 30 degrees C for 10 min, at which time the absorbance at 675 nm was measured. AO were calculated using the following formula: AO nmol/ml = 35 nmol/ml-(Es-Eb)/(Estd-Eb) x 35 nmol/ml (Es = sample abs., Eb = blank abs., Estd = standard abs.). Within-run precision for plasma AO was 2.3%. AO in plasma samples stored for 4 h at 4 degrees C was decreased by 1 nmol/ml. After 3 h at room temperature, AO was decreased by the same amount. Because this method measured ascorbic acid, alpha-tocopherol, glutathione peroxidase and quercetin as antioxidant compounds, we were able to measure antioxidant activity in human plasma. Our reference values were calculated from the volunteers group which consisted of 172 students and 82 soldiers. The reference intervals for plasma AO by this procedure were 15.4-20.9 nmol/ml.