[Measurement of superoxide radicals produced by human activated neutrophils by accumulation of stable nitroxide radicals detected by MR spectroscopy].
An important index of neutrophil function is the production of superoxide radicals (O2-) upon activation. Thus a development of a new adequate assay of O2- generation measurement is of great interest for phagocyte researchers. The present article considers the quantitative determination of O2- generation based on the interaction of O2- with 1-oxy-2,2,6,6-tetramethyl-4-oxypiperidine producing 4-oxo-2,2,6,6-piperidine-1-oxyl, detected by ESR. The kinetic curve of nitroxyl radical (NR) formation has a linear character. The NR formation rate after a short induction period (appr. 2 min.) approaches 3.3 X 10(-3) M/s, where cell concentration was 4 X 10(5) per ml. Hydroxylamine (3.8 mM) auto-oxidation rate is negligible as compared with activated neutrophils and is equal to 2 X 10(-9) M/s. Sensitivity NR to the presence of superoxide dismutase (SOD) came as evidence that NR formation is due O2- radicals. SOD (10(-7) M) inhibits NR formation by 90%. Hydroxylamine oxidation by O2- is an irreversible reaction--20-min incubation of activated neutrophils with NR do not influence NR concentration. The NR generation rate dependence upon the neutrophil concentration is linear in the cell concentration range from 4 X 10(5 up to 6 X 10(6) per ml. In this range a quantitative measurement of O2- production is suitable. The sensitivity of hydroxylamine assay is close to the sensitivity of chemiluminescent method, but specificity is higher, as SOD inhibits chemiluminescence only by 50%.
Osipov
A N
AN
Vartanian
L S
LS
Azizova
O A
OA
Vladimirov
Iu A
IuA
D005609:Free Radicals; D006801:Humans; D006898:Hydroxylamines / Q000032:analysis; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D009504:Neutrophils / Q000378:metabolism*; D009589:Nitrogen Oxides / Q000032:analysis*; D013482:Superoxide Dismutase / Q000032:analysis; D013481:Superoxides / Q000032:analysis*
D005609:Free Radicals; D006898:Hydroxylamines; D009589:Nitrogen Oxides; D013481:Superoxides; D013482:Superoxide Dismutase; C039900:nitroxyl
Izmerenie superoksidnykh radikalov, obrazuemykh aktivirovannymi neĭtrofilami cheloveka, po nakopleniiu stabil'nykh nitroksil'nykh radikalov, registriruemykh metodom EPR.