Reactive nitrogen species, including
nitrogen oxides (N(2)O(3) and N(2)O(4)),
peroxynitrite (ONOO(-)), and
nitryl chloride (NO(2)Cl), have been implicated as causes of
inflammation and
cancer. We studied reactions of secondary
amines with
peroxynitrite and found that both N-
nitrosamines and N-nitramines were formed.
Morpholine was more easily nitrosated by
peroxynitrite at alkaline pH than at neutral pH, whereas its nitration by
peroxynitrite was optimal at pH 8.5. The yield of nitrosomorpholine in this reaction was 3 times higher than that of nitromorpholine at alkaline pH, whereas 2 times more nitromorpholine than nitrosomorpholine was formed at pH <7.5. For the
morpholine-
peroxynitrite reaction, nitration was enhanced by low concentrations of
bicarbonate, but was inhibited by excess
bicarbonate. Nitrosation was inhibited by excess
bicarbonate. On this basis, we propose a
free radical mechanism, involving one-electron oxidation by
peroxynitrite of secondary
amines to form amino radicals (R(2)N(*)), which react with
nitric oxide ((*)NO) or
nitrogen dioxide ((*)NO(2)) to yield nitroso and nitro secondary
amines, respectively. Reaction of
morpholine with NO(*) and
superoxide anion (O(2)(*)(-)), which were concomitantly produced from
spermine NONOate and by the
xanthine oxidase systems, respectively, also yielded nitromorpholine, but its yield was <1% of that of nitrosomorpholine. NO(*) alone increased the extent of nitrosomorpholine formation in a dose-dependent manner, and concomitant production of O(2)(*)(-) inhibited its formation. Reactions of
morpholine with
nitrite plus HOCl or
nitrite plus H(2)O(2), with or without addition of
myeloperoxidase or
horseradish peroxidase, also yielded nitration and nitrosation products, in yields that depended on the reactants.
Tyrosine was nitrated easily by synthetic
peroxynitrite, by NaNO(2) plus H(2)O(2) with
myeloperoxidase, and by NaNO(2) plus H(2)O(2) under acidic conditions. Nitrated secondary
amines, e.g., N-nitroproline, could be identified as specific markers for endogenous nitration mediated by
reactive nitrogen species.