Determination of erythrocyte antioxidant capacity in haemodialysis patients using electron paramagnetic resonance.

Abstract

The increased oxidative stress of uraemia is caused both by an increased generation of oxygen-free radicals and a decrease of antioxidative forces. There are, however, conflicting data concerning disturbances of the radical-scavenging power of red blood cells (RBCs) in uraemic patients. The antioxidant capacities of the RBCs of 10 haemodialysis (HD) patients and 10 controls were examined after treatment with 0.324 mM tert-butylhydroperoxide (t-BOOH) in phosphate-buffered saline at 37 degrees C using electron paramagnetic resonance (EPR) with 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap and glutathione (GSH) regeneration as an indicator of hexose monophosphate shunt (HMPS) activity. EPR investigations were also done after pre-incubation with N-ethylmaleimide (NEM) to inhibit the GSH system. Furthermore, we determined the RBC redox state in 15 HD patients and 15 controls. There was no difference between HD patients and controls in the elimination of t-BOOH-generated free radicals in the RBCs. A more than 20-fold increase in radical concentration was observed after GSH trapping with NEM. In this case, we found a delayed decrease of the relative radical concentration in HD patients compared with controls with a significant difference after 7 min (2.2+/-0.26 vs 1.60+/-0.21; P=0.005) and after 10 min (1.82+/-0.41 vs 0.83+/-0.44; P=0.001). GSH regeneration via HMPS did not differ between the RBCs of HD patients (99.5+/-13.5 nmol/min x ml RBC) and those of the controls (94.2+/-16.9 nmol/min x ml RBC). There were no differences in the RBC concentrations of GSH, GSSG, NADP, NADPH, and in the GSH/GSSG and NADP/NADPH ratios between HD patients and controls. These data suggest a strong antioxidant potential in the GSH system of erythrocytes without any evidence of a disturbance in HD patients. The HMPS pathway also appears not to be impaired in the RBCs of HD patients. However, the slower radical elimination in the RBCs of HD patients after inhibition of GSH-depending radical scavengers as compared with controls indicates a defect in the antioxidant forces outside the GSH system, and could be one reason for the reduced lifespan of RBCs in HD patients.