Nitrating reactive nitric oxygen species transform acetaminophen to 3-nitroacetaminophen

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.