Myeloperoxidase Potentiates Nitric Oxide-mediated Nitrosation*

Nitrosation is an important reaction elicited by nitric oxide (NO). To better understand how nitrosation oc-curs in biological systems, we assessed the effect of myeloperoxidase (MPO), a mediator of inflammation, on nitrosation observed during NO autoxidation. Nitrosation of 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ; 10 (cid:1) M ) to 2-nitrosoamino-3-methylimidazo[4,5- f ]quinoline (N-NO-IQ) was monitored by HPLC. Using the NO donor spermine NONOate at pH 7.4, MPO potentiated N-NO-IQ formation. The minimum effective quantity of necessary components was 8.5 n M MPO, 0.25 (cid:1) M H 2 O 2 /min, and 0.024 (cid:1) M NO/min. Autoxidation was only detected at > 1.2 (cid:1) M NO/min. MPO potentiation was not affected by a 40-fold excess flux of H 2 O 2 over NO or less than a 2.4-fold excess flux of NO over H 2 O 2 . Potentiation was due to an 8.8-fold increased affinity of MPO-derived nitrosating species for IQ. Autoxidation was inhibited by azide, suggesting involvement of the nitrosonium ion, NO (cid:2) 4 OH. Authentic N-NO-IQ (98% pure) was treated in a similar manner to prepare standards. A recent study has verified the structure of these compounds (20). Samples were analyzed by HPLC on mobile phase 1 and 2. Statistical Analysis— Data are expressed as a mean (cid:7) S.E., and significant differences were evaluated using Student’s paired t test with p (cid:8) 0.05.

Nitric oxide (NO) 1 is an essential regulator for a variety of processes critical to normal functions in the cardiovascular, nervous, and immune systems (1). Impaired responses are observed with excessive production of NO in pathological conditions associated with chronic inflammation. Effects of NO can be divided into direct and indirect (2). Direct effects of NO are mediated by low nanomolar concentrations of NO and are illustrated by its binding to guanylate cyclase and eliciting numerous effects, including smooth muscle relaxation. In contrast, indirect effects occur at higher concentrations of NO and result from the reaction of NO with either oxygen (autoxidation) or superoxide to produce reactive nitrogen oxygen species (RNOS).
Indirect effects of NO elicited by RNOS include nitrosation, oxidation, and nitration reactions with numerous biological targets representing lipids, proteins, and DNA (2). This can cause lipid peroxidation, inhibition of enzymes, and deamination of DNA. Autoxidation of NO results in the formation of N 2 O 3 , which yields the nitrosonium ion, NO ϩ (3,4). The latter is a potent nitrosating agent. Autoxidation is slow and considered unlikely to occur in biological systems because NO can be rapidly inactivated. For example, NO is removed from the vascular compartment by near diffusion-limited interaction with erythrocyte oxyhemoglobin, yielding ferric hemoglobin and nitrate (5). However, significant amounts of S-, N-, and heme-nitros(yl)ation are detected in vivo, suggesting that modes of nitrosation other than by autoxidation must exist (6,7).
NO is a physiologic substrate for several mammalian peroxidases, including myeloperoxidase (MPO) (8). Direct spectroscopic and rapid kinetic studies support a facile reaction between peroxidases and NO (8,9). Peroxidases may play an important role in attenuating direct effects of NO. Organ chamber studies with preconstricted vascular and tracheal rings have demonstrated that catalytic amounts of peroxidase hydrolyzed NO (10). This prevented smooth muscle relaxation and NO-mediated ring dilation. A subsequent study further emphasized the role of MPO as a leukocyte-derived NO oxidase (11). The product of MPO metabolism of NO is thought to be NO ϩ (8). Surprisingly, little consideration has been given to the possible role of MPO-derived NO ϩ in biologically important nitrosations. Cellular nitrosation has been demonstrated to be distinct from that catalyzed by NO autoxidation in aqueous solution. Experiments, using an oxidant to convert NO directly to NO 2 ⅐ , demonstrated that this RNOS facilitates nitrosation by combining with NO to form N 2 O 3 (12). NO 2 ⅐ -mediated nitrosation was shown to be similar to that observed in cells. We have recently demonstrated RNOS nitrosation of the colon carcinogen 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), forming 2nitrosoamino-3-methylimidazo [4,5-f]quinoline (N-NO-IQ) (13). The latter binds DNA and is mutagenic. In this study, we have used IQ as a target for evaluating MPO potentiation of NOmediated nitros(yl)ation. In view of the potential close temporal and spatial association between NO formation (inducible nitric-oxide synthase), hydrogen peroxide (H 2 O 2 ) formation, and MPO during an inflammatory response, a better understanding of NO-mediated nitros(yl)ation may improve treatment and aid in prevention of inflammation-related diseases (i.e. certain cancers, atherosclerosis, and asthma).
Metabolism of IQ by RNOS-To assess NO-mediated nitrosation, [ 14 C]IQ (0.01 mM) was incubated in 100 mM potassium phosphate buffer, pH 7.4, containing 0 to 0.1 mM SpN, 0.1 mM DETAPAC, 1 mM glucose, 0 -20 milliunits/ml glucose oxidase, and 0 -150 nM MPO in a total volume of 0.1 ml for 40 min at 37°C. The reaction was started by the addition of SpN, and tubes capped. Blank values were obtained in the absence of SpN. The reactions were stopped by adding 10 mM ascorbic acid in dimethylformamide (0.05 ml). Samples were immediately frozen and kept at Ϫ70°C for analysis by HPLC mobile phase 1 (13).
To assess MPO nonnitrosative oxidation of IQ, [ 14 C]IQ (0.06 mM) was incubated in 100 mM potassium phosphate buffer, pH 5.5, containing 0.1 mM DETAPAC, 1 mM glucose, 15 milliunits/ml glucose oxidase, 0.3 mM NaNO 2 , and 85 nM MPO in a total volume of 0.1 ml for 60 min at 37°C (13). Reactions were started by the addition of [ 14 C]IQ. Blank values were obtained in the absence of glucose oxidase. The reactions were stopped by adding 10 mM ascorbic acid in dimethylformamide (0.05 ml). Samples were immediately frozen and kept at Ϫ70°C for analysis by HPLC mobile phase 1.
Preparation of Human Polymorphonuclear Neutrophils (PMNs)-Human blood from healthy donors was mixed with EDTA (0.2% final concentration) and immediately layered over an equal volume of neutrophil isolation medium from Robins Scientific Corp. (Sunnyvale, CA). Neutrophils were isolated by centrifugation using the manufacturer's specifications. Red blood cell contamination was eliminated by hypotonic lysis at 4°C. Cells were resuspended at 2 ϫ 10 6 cells/ml in Hanks' balanced salt solution with calcium, magnesium, and bicarbonate. All protocols were in accordance with institutional guidelines on research involving human subjects of either the St. Louis VA Medical Center or the University of Iowa. All participants gave written informed consent.
For experiments with MPO-deficient patients, blood was collected in lithium heparin tubes and shipped overnight for analysis the next day. A normal healthy control sample of blood was drawn and included in the shipment. Neutrophils were isolated by a two-step procedure. First red blood cells were removed by dextran sedimentation, M r 200,000 -300,000 (MP-Biomedicals, Irvine, CA). Neutrophils were isolated from the supernatant following Ficoll-Hypaque Plus (Amersham Biosciences) gradient centrifugation. Neutrophils were resuspended in Hanks' balanced salt solution with calcium, magnesium, and bicarbonate. Molecular characteristics of the two MPO-deficient patients have been reported (17,18).
Metabolism of IQ by Neutrophils-Neutrophils (0.3 ϫ 10 6 cells in 0.3 ml) were incubated with 0.01 mM [ 14 C]IQ in 12 ϫ 75-mm polypropylene tubes at 37°C for 36 min in Hanks' balanced salt solution containing calcium, magnesium, and bicarbonate without phenol red. Where indicated, 0.054 mM PMA, 1.2 M NO/min (0.05 mM SpN), 1 mM NaN 3 , and 0.1 mM NADH were added at 5, 6, 8, and 8 min, respectively, whereas 66 g/ml catalase and 66 g/ml SOD were present at zero time. Blank values were obtained in the absence of SpN. The reaction was stopped by placing on ice and freezing at Ϫ70°C. Samples were sonicated for 15 s three times, and 0.3 ml of dimethylformamide containing 2 mM ascorbic acid was added. Samples were spun at 1,500 ϫ g for 10 min. The supernatant was evaporated, and residue was dissolved in 0.1 ml of methanol/dimethylformamide (8:2) and analyzed by HPLC with mobile phase 1.
The oxidant burst response was measured for each PMN preparation (19). Superoxide generation was activated by the addition of PMA. Superoxide-specific reduction of cytochrome c was determined spectrophotometrically (⑀ 550 ϭ 21.1 mM Ϫ1 cm Ϫ1 ) and was inhibited by superoxide dismutase (10 g/ml). Values observed with cells in the absence of PMA were considered as blanks.
To assess the authenticity of neutrophil-derived N-NO-IQ, selected supernatants were pooled after HPLC analysis, evaporated to dryness, and converted to either 2-azido-3-methylimidazo[4,5-f]quinoline (2-N 3 -IQ) or 2-chloro-3-methylimidazo[4,5-f]quinoline (2-Cl-IQ) derivatives. For 2-N 3 -IQ, pH 2.0, ammonium formate was added along with 10 mM NaN 3 . After 60 min at 37°C, the pH was adjusted to pH 7.0 -7.4. For 2-Cl-IQ, the residue was dissolved in 0.18 ml of acetonitrile, and 0.02 ml of 1 N HCl was added for 30 min at 37°C and then neutralized with 1 N NH 4 OH. Authentic N-NO-IQ (98% pure) was treated in a similar manner to prepare standards. A recent study has verified the structure of these compounds (20). Samples were analyzed by HPLC on mobile phase 1 and 2.
Statistical Analysis-Data are expressed as a mean Ϯ S.E., and significant differences were evaluated using Student's paired t test with p Ͻ 0.05.

MPO Potentiation of NO-mediated Nitrosation-MPO
was evaluated to determine its effect on NO-mediated nitrosation of IQ ( Fig. 1). Previous studies have demonstrated significant transformation of IQ to a stable N-nitroso product, N-NO-IQ, during autoxidation of NO, using a high flux of NO (ϳ30 M NO/min, 0.1 mM diethylamine NONOate) (13). However, using a flux of NO more likely to occur during inflammation (2.4 M/min, 0.1 mM SpN), N-NO-IQ formation due to autoxidation (0.2 M) was near the limit of detection, 1.6% of the total radioactivity recovered by HPLC (Fig. 1, top). In the absence of NO, MPO (85 nM) and H 2 O 2 (10 M/min H 2 O 2 ) did not catalyze N-NO-IQ formation or metabolize IQ (Fig. 1, middle). When incubation mixtures contained NO, MPO, and H 2 O 2 , significant N-NO-IQ formation (3.1 M) was detected, 31% of the total radioactivity recovered by HPLC (Fig. 1, bottom) Nitrosation of IQ illustrated in Fig. 1 (top and bottom) was evaluated kinetically over a range of IQ concentrations (0.005-0.080 mM) (Fig. 6). Keeping the flux of NO constant (2.4 M/ min), N-NO-IQ formation was evaluated in the presence and absence of MPO (85 nM) and H 2 O 2 (10 M/min). The affinities of nitrosating species produced by NO for IQ were greater in the presence of MPO/H 2 O 2 (Ϫ1/X int ϭ 21 ϩ 2 M) than with NO alone by autoxidation (Ϫ1/X int ϭ 189 ϩ 3 M). By contrast at infinite concentrations of IQ, the maximal rates of N-NO-IQ formation were similar with MPO/H 2 O 2 (1/Y int ϭ 24 ϩ 3 pmol/ min) and autoxidation (1/Y int ϭ 28 ϩ 1 pmol/min). Thus, potentiation of NO-mediated IQ nitrosation is due to increased affinity of MPO-derived RNOS for IQ.
Inhibition of NO and MPO Potentiation of NO Nitrosation-To assess N-NO-IQ formation mediated by NO autoxidation, a high NO flux (9.6 M/min) was necessary to test different agents ( Table I). Autoxidation of IQ was not significantly altered by 100 mM NaCl, 33 g/ml catalase, or 33 g/ml SOD. Inhibition of MPO Nonnitrosative Oxidation-To help interpret the effects of test agents in Table I, the same test agents were used to evaluate MPO nonnitrosative oxidation of IQ (Table II). This oxidation of IQ at pH 5.5 requires not only H 2 O 2 , but also NO 2 Ϫ (13). A previous study identified these    (Table I) and oxidizes IQ with NO 2 Ϫ , an end product of NO metabolism (Table II).

TABLE I Effect of test agents on N-NO-IQ formation by NO with and without MPO
NO-mediated Nitrosation with Human PMNs-To assess the significance of MPO potentiation of NO-mediated IQ nitrosation, N-NO-IQ formation by human PMNs was assessed (Table  III). Cells were incubated with 0.01 mM IQ and 1.2 M NO/min (Fig. 7, top). In the absence of PMA, the amount of N-NO-IQ formed was similar to that observed in the absence of cells (not shown). PMA increased N-NO-IQ formation 2.7-fold. This increase was prevented by 66 g/ml catalase. Values observed with PMA were further increased by the presence of 66 g/ml SOD (2-fold) (Fig. 7, middle). Catalase and NADH, but not azide, suppressed the increase observed with PMA and SOD. To assess the authenticity of N-NO-IQ, neutrophil-derived N-NO-IQ was converted to 2-N 3 -IQ by pH 2.0/10 mM NaN 3 treatment. As illustrated in Fig. 7 (bottom), this treatment resulted in the disappearance of the N-NO-IQ peak observed in Fig. 7 (middle panel) at 10.2 min and the appearance of a new peak at 15.5 min, 2-N 3 -IQ. Synthetic 2-N 3 -IQ standard also eluted at 15.5 min. The yield of 2-N 3 -IQ was 52%. The elution time of neutrophil-derived and synthetic 2-N 3 -IQ was identical on a different HPLC solvent system. In addition, treatment of neutrophil-derived N-NO-IQ with 1 N HCl produced 2-Cl-IQ. Neutrophil-derived and synthetic 2-Cl-IQ were shown to have identical elution times on two different HPLC solvent systems. This confirms the formation of N-NO-IQ by PMA-stimulated neutrophils exposed to a constant low flux of NO. Results with human neutrophils are consistent with MPO potentiation of NO-mediated IQ nitrosation.
To more directly demonstrate a catalytic role for MPO in IQ nitrosation by PMNs, two unrelated patients with MPO-deficient PMNs were evaluated. Normal PMNs incubated with 0.01 mM IQ and 1.2 M NO/min for 36 min produced 26 Ϯ 3 pmol of N-NO-IQ. In maximally stimulated neutrophils, PMA ϩ SOD produced significantly more N-NO-IQ, 42 Ϯ 3 pmol (p Ͻ 0.01). This increase in N-NO-IQ formation was completely inhibited by catalase. As demonstrated in Fig. 7 (bottom), the N-NO-IQ HPLC peak was converted to 2-N 3 -IQ by incubation of PMNderived material under acidic conditions with 10 mM NaN 3 . In the MPO-deficient PMNs, N-NO-IQ production observed for one patient was 27 Ϯ 1 and 26 Ϯ 2 pmol for neutrophils and neutrophils with PMA ϩ SOD, respectively. For the second patient, N-NO-IQ production was 26 Ϯ 2 and 30 Ϯ 2 pmol for neutrophils and neutrophils with PMA ϩ SOD, respectively. The oxidant bursts observed with PMNs from the normal control and MPO-deficient patients were similar. The inability of MPO-deficient PMNs to increase N-NO-IQ formation in response to PMA ϩ SOD treatment is consistent with a role for MPO in PMN nitrosation of IQ. DISCUSSION This is the first study to demonstrate MPO potentiation of NO-mediated nitrosation. MPO did not metabolize IQ in the absence of NO. With NO, MPO potentiates nitrosation of IQ observed with the NO donor SpN, forming N-NO-IQ. The latter is the only product observed. To simulate the in vivo situation, H 2 O 2 was generated in situ. Potentiation of N-NO-IQ formation was observed over a range of MPO, NO, and H 2 O 2 concentrations likely to occur during an inflammatory response. MPO altered the kinetics of N-NO-IQ formation by producing nitrosating species with an 8.8-fold higher affinity for IQ than those produced by autoxidation. Potentiation by MPO was not affected by a 40-fold excess flux of H 2 O 2 over NO or less than a 2.4-fold excess flux of NO over H 2 O 2 . To further relate these observations to the in vivo situation, stimulated human PMNs demonstrated increased N-NO-IQ formation. This increase was not detected with PMNs from MPO-deficient patients, consistent with a role for MPO in PMN nitrosation of IQ. The authenticity of neutrophil-derived N-NO-IQ was confirmed by its conversion to 2-chloro and 2-azido derivatives. Thus, MPO provides an alternative pathway for nitrosation that may help explain part of the significant amount of nitrosation occurring in vivo (6,7).
RNOS have a dramatic effect on MPO metabolism of IQ. In the absence of NO or NO 2 Ϫ , MPO metabolism of IQ was not detected. NO 2 Ϫ , a stable end product of NO autoxidation, is converted to NO 2 ⅐ by MPO (21). NO 2 ⅐ has a high reduction potential and oxidizes dopamine to dopamine semiquinone (22). NO 2 ⅐ may also oxidize IQ to IQ ⅐ , which may subsequently combine with NO 2 ⅐ , forming nitrated IQ products, or with IQ ⅐ , forming IQ dimer (13). These products are reported in Table II. In contrast with NO, MPO potentiates IQ nitrosation, forming N-NO-IQ. IQ is useful for demonstrating both nitration and nitrosation pathways catalyzed by MPO.
A variety of test agents were used to evaluate IQ nitrosation during NO autoxidation (Table I). This nitrosation is thought to be elicited by NO ϩ following formation of N 2 O 3 (3). Azide has   been shown to react with NO ϩ preventing nitrosation (23) and has been used to distinguish nitrosation from oxidative nitrosylation reactions (24). Using the latter criteria, 70% of N-NO-IQ formation by autoxidation was due to nitrosation. NADH can trap NO 2 ⅐ (25). Inhibition by NADH suggests that N-NO-IQ formation might involve NO 2 ⅐ or a NO 2 ⅐ -like species. NO 2 ⅐ could react with NO, facilitating formation of N 2 O 3 . The latter would then form NO ϩ . This proposed pathway is consistent with inhibition by both azide and NADH, as reported in Table I. The small decrease observed with NaCl is consistent with the reported reaction of biologically relevant nucleophiles with NO ϩ (26). Whereas inhibition by ascorbic acid might be attributed to its antioxidant properties on reactive intermediates, it also readily reduces MPO compounds I and II. DMPO inhibition suggests radical involvement (27). The lack of significant inhibition by catalase or SOD is consistent with neither H 2 O 2 nor superoxide playing a significant role. Thus, N-NO-IQ formation during autoxidation of NO is consistent with involvement of NO ϩ and NO 2 ⅐ or a NO 2 ⅐ -like species. The generation of these RNOS during autoxidation has been debated (4,12,23). Although individual test agents may have more than one action and may not provide unequivocal proof of the RNOS involved, they do characterize nitrosation attributed to autoxidation and allow comparisons with that process.
MPO potentiation of N-NO-IQ formation was distinct from that observed by autoxidation. Azide did not inhibit N-NO-IQ formation, suggesting that NO ϩ was not involved. Consistent with MPO-catalyzed metabolism, catalase completely inhibited N-NO-IQ formation. NADH caused complete inhibition, suggesting formation of a NO 2 ⅐ -like RNOS. Effects of ascorbic acid and DMPO were consistent with their antioxidant/reducing substrate and radical trapping effects, respectively. The lack of effect of SOD demonstrated that superoxide is not involved. Decreased N-NO-IQ formation at high levels of NO (Fig. 5) could be due to ligation of NO to the distal heme, which prevents access of H 2 O 2 to the catalytic site (9). Since NO ϩ has been proposed as the product of MPO metabolism of NO (8), results with azide and NADH were surprising. The lack of azide inhibition of nitrosation has been previously reported and attributed to oxidative nitrosylation (24). This pathway could involve a RNOS, such as NO 2 ⅐ , oxidizing IQ to IQ ⅐ , with the latter combining with NO to form N-NO-IQ. The reaction elicited by the RNOS derived from MPO oxidation of NO is consistent with oxidative nitrosylation rather than nitrosation.
MPO nonnitrosative oxidation of IQ (Table II) was different from that observed in the presence of NO (Table I). With NO 2 Ϫ present, 0.3 mM NaN 3 appears to be functioning as a heme poison and completely inhibits MPO metabolism of IQ. NO 2 Ϫ is a poor substrate for MPO (28). In contrast, NO is a good substrate for MPO and can bind the distal heme at the active site (8,9). This may help explain why azide was a potent inhibitor of NO 2 Ϫ , but not NO oxidation of IQ. MPO converts NO 2 Ϫ to NO 2 ⅐ (21). Whereas trapping of NO 2 ⅐ by NADH could explain reduced IQ nitration, this reduction was modest compared with that observed with N-NO-IQ formation. NADH is also a substrate for peroxidases and could function as a competitive inhibitor. However, since NO is a better substrate than NO 2 Ϫ , one would expect more inhibition by NADH of the NO 2 Ϫ than the NO reaction. The nitration of IQ reported here should be consistent with that observed for MPO-catalyzed formation of 3-nitrotyrosine or 3-nitroacetaminophen (29,30). The nitrating, chlorinating, and oxidizing activities of MPO are reported to be inhibited by 1 mM azide (31). A physiological concentration of Cl Ϫ significantly reduced the formation of the nitrated products more than 60%. Consistent with a previous study (8), this concentration of Cl Ϫ did not alter MPO metabolism of NO (Table I). The lack of Cl Ϫ inhibition of NO metabolism was previously attributed to higher substrate affinity of NO for MPO than Cl Ϫ (8,9). Effects of ascorbic acid and DMPO were consistent with their antioxidant/reducing substrate and radical trapping effects, respectively. Thus, the relative substrate and ligand affinities of NO, NO 2 Ϫ , NaN 3 , and NaCl for MPO help to explain the diverse metabolite profiles observed. The diffusion-limited reaction of NO with superoxide to form peroxynitrite is thought to play an important role in nitrosation and oxidative nitrosylation (24,32). Nearly equimolar fluxes of each reactant were shown to yield maximum nitrosation of the amines diaminonaphthalene and diaminofluorescein. An excess of one reactant over the other precipitated a decrease in nitrosation. This was attributed to secondary reactions occurring directly between peroxynitrite and excess NO or superoxide. Product formation was influenced by SOD, but not catalase. NO 2 ⅐ , a metabolite of peroxynitrite, was proposed to facilitate nitrosation by combining with NO to form N 2 O 3 , which yields NO ϩ , an azide-sensitive product. In addition, NO 2 ⅐ oxidized these amines to radicals, which then combine with NO forming the same product by oxidative nitrosylation. The latter reaction was not azide-sensitive but would be expected to be inhibited by NADH. Whereas potentiation of NO-mediated nitros(yl)ation by in situ generated peroxynitrite exhibits some differences in sensitivities to inhibitors from that observed with MPO, similar RNOS, such as NO 2 ⅐ or a NO 2 ⅐ -like species, may be involved in both reactions. MPO can potentiate NO consumption in the presence of a superoxide-generating system (8). Future studies will assess potentiation of N-NO-IQ formation by fluxes of NO and superoxide.
Human PMNs were used to simulate the in vivo condition. In the presence of SpN, PMNs produced increased amounts of N-NO-IQ following stimulation with PMA. The latter results in the release of MPO and causes an oxidant burst that produces superoxide, which dismutates to H 2 O 2 (33). This oxidant burst is mediated by NADPH-oxidase reduction of molecular oxygen. Maximum N-NO-IQ formation was observed with SOD, suggesting that peroxynitrite was not involved. The increase observed with SOD could be due to decreased inhibition of MPO by superoxide and/or increased presence of H 2 O 2 (33). Catalase, by hydrolyzing H 2 O 2 , reduced MPO activity and reduced N-NO-IQ formation. The lack of azide inhibition and inhibition by NADH is similar to that observed with MPO in Table I and consistent with oxidative nitrosylation. Studies assessing nitration and chlorination by PMNs have also demonstrated increased activity with SOD. However, nitration and chlorination were inhibited by catalase and azide (28,34). N-NO-IQ is the only major product formed by stimulated PMNs, accounting for greater than 90% of the increased metabolism of IQ observed with PMA-stimulated PMNs treated with SOD. This increase in N-NO-IQ formation observed with PMA ϩ SOD was not detected with PMNs from two unrelated MPO-deficient patients that exhibited a normal oxidant burst. Thus, MPO potentiates IQ nitrosylation by stimulated human PMNs exposed to NO.
Inflammatory bowel disease is a chronic inflammatory disease associated with marked mucosal infiltration of macrophages, lymphocytes, and neutrophils in which high levels of iNOS and 3-nitrotyrosine, a marker of RNOS, are detected (35)(36)(37). The temporal and spatial association necessary for the various components (MPO, H 2 O 2 , and NO) to potentiate NOmediated nitrosation probably exists in colons of inflammatory bowel disease patients eating well done red meat (IQ) (38). Previous studies have demonstrated that MPO activates N-NO-IQ to bind DNA (13). Thus, MPO could be intimately involved in IQ initiation of colon cancer by participating in both the formation of N-NO-IQ and its activation to bind DNA.
Nitrosation due to autoxidation of NO is a slow process (Fig.  1, upper panel) and was only detected at fluxes of NO Ն1.2 M/min (Fig. 5). The autoxidation of NO is inversely related to its concentration and at concentrations that might occur during an inflammatory response, long half-lives of NO are expected.
The half-life of 1 and 10 M NO at 37°C and pH 7.4 can be calculated to be ϳ10 and 1 min, respectively (4). MPO potentiation of N-NO-IQ formation was detected at NO fluxes as low as 0.024 M/min (Fig. 4). In addition to MPO, NO is also a substrate for eosinophil peroxidase, lactoperoxidase, and prostaglandin H synthase (8,10,39). These peroxidases would also be expected to potentiate NO-mediated nitrosation in inflammatory conditions such as inflammatory bowel disease, atherosclerosis, and asthma. Preliminary results in our laboratory have demonstrated ovine prostaglandin H synthase potentiation of N-NO-IQ formation. Indirect effects of NO attributed to autoxidation (2) might be due to peroxidases. Whereas nitrosation is thought to occur at sites of NO generation (2,40), our results suggest that this highly diffusible stable gas initiates nitrosation at sites of neutrophil infiltration. This information provides insight for therapeutic intervention and prevention of certain inflammatory diseases.