Peroxynitrite formation from activated human leukocytes.

We showed direct evidence of peroxynitrite formation from polymorphonuclear cells (PMN) with the nitration of 4-hydroxyphenylacetic acid (HPA) to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA). 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). NO2HPA was detected under PMA stimulation only in the presence of myeloperoxidase inhibitor. NO2HPA was eliminated by N-monomethyl-L-arginine (100 microM). The inhibition of myeloperoxidase appears to be essential to demonstrate the production of NO2HPA since myeloperoxidase itself or its product, hypochlorite, reacted with peroxynitrite and hampered the formation of NO2HPA.