Assay for oxidative stress injury by detection of luminol-enhanced chemiluminescence in a freshly obtained blood sample: A study to follow the time course of oxidative injury

To evaluate occurrence of oxidative stress in circulating blood, we developed standard methods to assess (1) granulocytes status as a source of reactive oxygen species (ROS) and (2) lipid peroxidation (LPO). A simplified and highly sensitive assay was developed by utilizing the chemiluminescence (CL) from luminol oxidized by ROS. 1. The CL, from 300 μl medium containing 1% blood, 10 μg/ml luminol and 0.025 μg/ml phorbol myristate acetate, well reflected the primed granulocyte status induced by in vitro contact with lipopolysaccharide (LPS). This CL was weakened slightly by superoxide dismutase and catalase, but markedly decreased by sodium azide. 2. We determined the optimal conditions for the t-butyl hydroperoxide (t-BuOOH)- stimulated CL method to evaluate plasma LPO in experiments on rat plasma added with phosphatidylethanolamine hydroperoxide (PEOOH). The CL from 300 μl medium containing 6.67% plasma, 10 μg/ml luminol and 5 μ mol/ml t- BuOOH was proportional to the added PEOOH amount. The integrated CL of the plasma with 0-60 nmol of PEOOH gave values of 8.280-14.213 x 10⁶ counts/60 min/tube. 3. Only 100 μl of freshly drawn blood was enough for the two CL methods to detect the generation of ROS and the occurrence of LPO. These CL methods enabled the determination of the time course of oxidative stress occurrence in circulating blood of rats treated with 5 mg/kg LPS, i.p.