Peroxynitrite formation from activated human leukocytes

We showed direct evidence of peroxynitrite formation from polymorphonuclear cells (PMN) with the nitration of 4-hydroxpphenylacetic acid (HPA) to 4-hydroxy-3-nitrophenylacetic acid (NO(2)HPA). Human PMN from healthy volunteers was stimulated with phorbol-12-myristate-13-acetate (PMA, 10 ng/ml) at 37 degrees C in 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid-buffered Hank' s balanced salt solution (pH 7.4) with HPA (1 mM). NO(2)HPA was detected under PMA stimulation only in the presence of myeloperoxidase inhibitor. NO(2)HPA was eliminated by N-monomethyl-L-arginine (100 mu M). The inhibition of myeloperoxidase appears to be essential to demonstrate the production of NO(2)HPA since myeloperoxidase itself or its product, hypochlorite, reacted with peroxynitrite and hampered the formation of NO(2)HPA. (C) 1996 Academic Press, Inc.