Nitrating reactive nitric oxygen species (RNOS) elicit many of the deleteri
ous effects of the inflammatory response. Their high reactivity and short h
alf-life make RNOS analysis difficult. Reaction of acetaminophen (APAP) wit
h RNOS generated by various conditions was evaluated by HPLC. When [C-14]AP
AP was incubated at pH 7.4, the same new product (3NAP) was produced by at
least three separate pathways represented by the following conditions: myel
operoxidase oxidation of NO2-, NO2Cl, and ONOO- or Sin-1. Diethylamine NONO
and spermine NONO did not convert APAP to 3NAP. 3NAP was stable at pH 5, 7
.4, or 9, and at pH 7.4 with ONOO-, spermine NONO, Sin-1, or H2O2. HOCl tra
nsformed 3NAP, which was prevented by APAP, ascorbic acid, taurine, or NO2-
. ONOO--derived 3NAP was identified by H-1 NMR as 3-nitroacetaminophen or 3
-nitro-N-acetyl-p-aminophenol, and the product mass was verified by EI/ESI
mass spectrometry. Human polymorphonuclear neutrophils incubated with [C-14
]APAP and stimulated with beta-phorbol 12-myristate 13-acetate produced 3NA
P in the presence of NO2-. Neutrophil 3NAP formation was verified by mass s
pectrometry and was consistent with myeloperoxidase oxidation of NO2-. Sper
mine NONO supported 3NAP formation by stimulated cells in the absence of NO
2-. Results demonstrate that 3NAP is a product of nitrating RNOS generated
by at least three separate pathways and may be a biomarker for nitrating me
diators of inflammation.