Hydroxyl free-radical spin-adduct in rat brain synaptosomes Observations on the reduction of the nitroxide

Understanding the prevalence and action of hydroxyl free-radicals species in biological systems, is improved by spin-trapping techniques. The hydroxyl free-radical rapidly adds to the spin-trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) to form a relatively stable nitroxyl free-radical spin-adduct (DMPO-OH), but it was found that DMPO-OH becomes diamagnetic, presumably by chemical reduction, when it is added to rat brain synaptosomes. DMPO-OH reduction by synaptosomes was a time-dependent process, starting immediately and continuing up to 20 min or more, when almost all the spin-adduct is reduced. During synaptosomal reduction, the electron paramagnetic resonance (EPR) high field line of DMPO-OH is broadened indicating restrictions on the motion of the nitroxide. This wasnot due to simple absorption of DMPO-OH to proteins. Experiments with the spin-adduct in glycerol at low temperatures showed that DMPO-OH experienced, during synaptosomal reduction, restricted motion having a correlation time of about 3.3·10^?9 s. This value would be expected if DMPO-OH was near the polar region of the hydrocarbon layer of a membrane bilayer. Synaptosomal membrane transport of DMPO-OH was tested by competition studies, since it was presumed that this nitroxide was transported and then reduced inside the synaptosomes. Several compounds tested, including amino acids, neurotransmitters, food dyes, and many others, that may have acted competitively in transport were not effective in slowing down reduction of DMPO-OH. However, it was found that azide, cyanide and heating of the synaptosomes markedly decreased the rate of synaptosomal reduction of DMPO-OH. Azide at 50μM manifested one-half of its maximal inhibitory effect. Experiments with 1-octanol to obtain a measure of the membrane partition of DMPO-OH demonstrated that the octanol/water distribution was 1.83. Experiments with glass microfibre filtration to determine synaptosomal binding revealed that DMPO, as well as apparently DMPO-OH, were not specifically retained by synaptosomes. All of these results combined with past work on nitroxide reduction by biological systems, suggest that DMPO-OH reduction by synaptosomes is due to its interaction with and reduction by electron transport carrier of mitochondria within the synaptosomes.