NITRIC-OXIDE INTERACTION WITH LACTOFERRIN AND ITS PRODUCTION BY MACROPHAGE CELLS STUDIED BY EPR AND SPIN-TRAPPING

The production of nitrate (NO3-) and nitrite (NO2-) from macrophage-de rived NO. was studied using EPR and spin trapping. The formation of NO 3- was determined via EPR in reactions involving the iron-binding prot ein, lactoferrin. The formation of NO2- was determined via EPR/spin tr apping in the reaction between NO2- and H2O2. Dissolved nitric oxide ( NO.) was reacted with lactoferrin yielding an EPR spectrum (77-degrees -K) different from the normal EPR spectrum obtained for lactoferrin, s uggesting that NO. interacts with the ferric ions bound to lactoferrin forming a ferric-nitrosyl type complex. The EPR spectrum (77-degrees- K) of this ferric-nitrosyl type complex was also observed in the super natant fluid of macrophage cell suspensions following their stimulatio n with lipopolysaccharide (LPS). During LPS stimulation of macrophages , these cells generate NO. which in turn produces NO3- and NO2-. The f erric-nitrosyl type complex is formed in a reaction mixture containing apolactoferrin and bicarbonate following the reaction of Fe+2 with NO 3-, generated from macrophage-derived NO., to produce Fe+3 and NO.. Fu rthermore, in an acidic medium, NO2- reacts with H2O2 forming peroxyni trous acid (HOONO) which rapidly decomposes into hydroxyl radicals (.O H) and the nitrogen dioxide (NO2.) radical. In the supernatant fluid o f LPS-stimulated macrophage suspensions, the production of OH was veri fied by spin trapping using 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) as the spin trap and ethanol as the .OH scavenger. The EPR spectra corre sponding to the DMPO-OH and the DMPO-hydroxyethyl adducts were identif ied. These results suggest that the peroxynitrous acid decomposes via the formation of .OH and NO2. and that NO2- was formed from macrophage -derived NO..