Hypoxia-stimulated reduction of doxyl stearic acids in human red blood cells. Role of hemoglobin.
Nitroxide free radicals are under active investigation for their potential use as metabolically responsive contrast agents in electron paramagnetic resonance and nuclear magnetic resonance imaging. The metabolism in human red blood cells of lipid-soluble nitroxides, doxyl stearic acids (DSA), has been investigated. We observed that under normoxia DSA were stable in red blood cells for at least 2 h, but hypoxia stimulated spin label reduction. Complete signal recovery after air or ferricyanide oxidation suggested the formation of hydroxylamine during hypoxia. DSA reduction was found to be dependent upon the position of the nitroxide ring in the fatty acid chain with the reduction rate higher when the -NO degree of the doxyl ring was closer to the fatty acid carboxylic end. The reduction kinetics of DSA with the doxyl ring nearest to the carboxylic end (5DSA) was bifasic. A rapid reduction of about half of the 5DSA was observed in the first hour and, thereafter, a slow reduction process become predominant. The slope of the slow reduction abruptly decreased below 5 microM, thus suggesting a concentration-dependent membrane-cytoplasm translocation of 5DSA. The reducing activity of the red blood cell (RBC) was completely recovered in the cell lysate. Under hypoxia, purified hemoglobin and myoglobin reduced 5DSA and a complete recovery of the signal was obtained after air reoxidation. Globin did not reduce 5DSA, while methemoglobin showed only a small reduction of 5DSA, thus suggesting that ferrous-heme was involved in the hypoxic reduction of DSA. both DSA localization and the characteristics of intracellular reductant (hemoglobin) are responsible for the high stability of DSA in the RBC.
Minetti
M
M
Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, Roma, Italy.
Scorza
G
G
D003497:Cyclic N-Oxides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000378:metabolism*; D005609:Free Radicals; D006207:Half-Life; D006454:Hemoglobins / Q000502:physiology*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction
D003497:Cyclic N-Oxides; D005609:Free Radicals; D006454:Hemoglobins; C037404:12-doxylstearic acid; C005072:5-doxylstearic acid