The reduction of nitroxide spin label as a probe of human blood antioxidant properties

The kinetics of reduction of the radical R^•, 5-dimethyla minonaphthalene-1-sulfonyl-4-amino-2,2,6,6-tetramethyl- 1-piperidine-oxyl, by blood and its components were studied using the EPR technique. The results demonstrate that R^• is adsorbed to the outer surface of the membrane and does not penetrate into the erythrocytes. A series of control experiments in PBS demonstrate that ascorbate is the only natural reducing agent that reacts with R^•. The observed first order rate of disappearance of the nitroxide radical, κ, is: κ_blood > κ_eryth > κ_plasma and κ_blood ≅ κ_eryth + κ_plasma. The results demonstrate that: a. The erythrocytes catalyze the reduction of R^• by ascorbate. b. The rate of reduction of the radical is high though it does not penetrate the cells. c. In human erythrocytes there is an efficient electron transfer route through the cell membrane. d. The study points out that R^• is a suitable spin label for measuring the reduction kinetics and antioxidant capacity in blood as expressed by reduction by ascorbate.