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

Background. 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....

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Published in:Nephrology, dialysis, transplantation dialysis, transplantation, 2001-11, Vol.16 (11), p.2166-2171
Main Authors: Klemm, Andree, Voigt, Christine, Friedrich, Manfred, Fünfstück, Reinhard, Sperschneider, Heide, Jäger, Ernst‐G., Stein, Günter
Format: Article
Language:English
Subjects:
Adult
Aged
anaemia
Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy
antioxidant capacity
Antioxidants - metabolism
Biological and medical sciences
electron paramagnetic resonance spectroscopy
Electron Spin Resonance Spectroscopy
Emergency and intensive care: renal failure. Dialysis management
Erythrocytes - metabolism
Feasibility Studies
Female
Free Radical Scavengers - antagonists & inhibitors
Glutathione - physiology
haemodialysis
Humans
Intensive care medicine
Male
Medical sciences
Middle Aged
oxygen radicals
Pentose Phosphate Pathway - physiology
Reactive Oxygen Species - blood
red blood cells
Reference Values
Renal Dialysis
tert-Butylhydroperoxide - pharmacology
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Summary:Background. 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. Methods. 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°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. Results. 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×ml RBC) and those of the controls (94.2±16.9 nmol/min×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. Conclusions. 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.
ISSN:0931-0509
1460-2385
DOI:10.1093/ndt/16.11.2166