Tp. Bradshaw et al., IDENTIFICATION OF FREE-RADICALS PRODUCED IN RAT ERYTHROCYTES EXPOSED TO HEMOLYTIC CONCENTRATIONS OF PHENYLHYDROXYLAMINE, Free radical biology & medicine, 18(2), 1995, pp. 279-285
Previous studies have shown that incubation of rat red blood cells in
vitro with phenylhydroxylamine (50-300 mu M) induces rapid splenic seq
uestration of the red cells on reintroduction to isologous rats. EPR a
nd the spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), w
ere utilized to determine if free radical species could be identified
under these experimental conditions. Hemolytic concentrations of pheny
lhydroxylamine, in the presence of DMPO and 5-20% lysed or intact rat
erythrocyte suspensions, gave rise to a four-line (1:2:2:1) EPR spectr
um. No signal was obtained if phenylhydroxylamine, DMPO, or red cells
was omitted. Comparison of the phenylhydroxylamine-induced signal with
authentic hydroxyl radical- and GSH thiyl radical-DMPO standard adduc
t signals identified the phenylhydroxylamine-induced species as a GSH
thiyl free radical. Removal of GSH from a red cell lysate abolished th
e GSH thiyl radical signal without the appearance of any other signal,
while addition of exogenous GSH resulted in its return. When erythroc
ytes were exposed to concentrations of phenylhydroxylamine greater tha
n or equal to 200 mu M, a time-dependent transition of the GSH thiyl r
adical signal to a hemoglobin thiyl radical signal was observed. The d
ata are consistent with the postulate that thiyl radical species, gene
rated from the interaction of phenylhydroxylamine and oxyhemoglobin, p
lay a key role in the development of hemolytic injury to the rat red c
ell.