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Circulation Research. 1999;85:950-958

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(Circulation Research. 1999;85:950.)
© 1999 American Heart Association, Inc.

Cellular Biology

Formation of Nitric Oxide–Derived Oxidants by Myeloperoxidase in Monocytes

Pathways for Monocyte-Mediated Protein Nitration and Lipid Peroxidation In Vivo

Stanley L. Hazen, Renliang Zhang, Zhongzhou Shen, Weijia Wu, Eugene A. Podrez, Jennifer C. MacPherson, David Schmitt, Shome N. Mitra, Chaitali Mukhopadhyay, Yonghong Chen, Peter A. Cohen, Henry F. Hoff, Husam M. Abu-Soud

From the Department of Cell Biology (S.L.H., R.Z., Z.S., E.A.P., J.C.M., D.S., S.N.M., C.M., H.F.H., H.M.A.-S.), the Department of Cardiology (S.L.H.), and the Center for Surgery Research (A.C.), Cleveland Clinic Foundation, Cleveland, Ohio, and the Chemistry Department (S.L.H., W.W., Y.C., H.F.H.), Cleveland State University, Cleveland, Ohio.

Correspondence to Stanley L. Hazen, Cleveland Clinic Foundation, Lerner Research Institute, Department of Cell Biology, 9500 Euclid Ave, NC-10, Cleveland, OH 44195. E-mail hazens{at}ccf.org


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO2-), a major end product of nitric oxide (•NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography–mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO2-; occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous •NO generator PAPA NONOate (Z-[N-{3-aminopropyl}-N-{n-propyl}amino]diazen-1-ium-1,2-diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of •NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of •NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadienoic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating •NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.


Key Words: nitrotyrosine • atherosclerosis • lipid peroxidation • nitric oxide


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Oxidative damage of LDL is thought to be a critical step in atherogenesis.1 2 3 4 Accordingly, both the ability of cells present in the artery wall to accelerate LDL oxidation and the mechanisms through which cells generate reactive intermediates are currently of great interest. One pathway for LDL modification in vivo is through reactive nitrogen species. Immunohistochemical studies demonstrate that nitrotyrosine, a global marker of protein damage by nitrating intermediates, is increased in human atherosclerotic intima.5 Moreover, mass spectrometric studies demonstrate that the nitrotyrosine content of LDL recovered from human atherosclerotic aortas is nearly 100-fold higher than that observed in circulating LDL from healthy donors.6 We recently demonstrated that myeloperoxidase (MPO), a heme protein enriched in human atherosclerotic lesions, can generate nitrating intermediates that convert LDL into a high-uptake form for macrophages.7 However, characterization of the role of MPO versus other pathways in monocytes to generate reactive nitrogen species has not yet been examined. Despite a wealth of evidence that reactive nitrogen species contribute to protein and lipoprotein modification during vascular disease, neither the cellular sources of nitrating intermediates nor the reaction pathways for their generation in vivo are established.

Multiple cells present in the vasculature (eg, macrophages, endothelial cells) are capable of generating nitric oxide (•NO), a long-lived free radical with potent biological activities.8 •NO is a weak reducing agent and does not react with most biological targets; however, a variety of reactive nitrogen species derived from •NO are powerful oxidants, and they may contribute to oxidative damage.9 10 11 The cellular sources of reactive nitrogen species in atherosclerotic tissues is not established. It is generally assumed, however, that monocytes play a role in their production. Monocytes can be stimulated with various cytokines and vasoactive hormones to produce •NO by upregulating expression of the inducible isoform of nitric oxide synthase (NOS) (reviewed in Reference 1212 ). Monocytes also undergo a respiratory burst after activation by a variety of agonists, producing reduced oxygen species such as superoxide (O2•-) and hydrogen peroxide (H2O2).13 14 Hence, it has been concluded that monocytes may generate peroxynitrite (ONOO-), a potent nitrating oxidant formed by the interaction of •NO and O2•-.5 6 9

Despite the fact that monocytes are thought to play an essential role in both atherogenesis15 and the generation of reactive nitrogen species in vascular tissues,1 2 3 4 16 17 the chemical pathways available to monocytes for generating •NO-derived oxidants have not yet been directly examined. Indeed, no reports to date demonstrate that monocytes can promote the nitration of free or protein-bound tyrosine residues. Moreover, enhanced •NO production by peritoneal macrophages16 or from an exogenous source18 19 has been reported to inhibit rather than augment the extent of LDL lipid peroxidation. Thus, direct examination of the pathways available to monocytes for generating reactive nitrogen species and the characterization of the conditions under which they participate in protein and lipid modification must be addressed.

Studies thus far into the pathways available for LDL modification by reactive nitrogen species have relied primarily on in vitro chemical systems, neutrophils, and exogenous •NO generating systems. On the basis of these studies, at least 3 distinct pathways for generating reactive nitrogen species have been suggested. The first involves formation of ONOO- through the interaction of •NO and O2•-.9 The exposure of LDL to ONOO- results in the nitration of apolipoprotein B-100 (apo B-100) tyrosine residues,6 the initiation of lipid peroxidation,20 21 and the depletion of lipid-soluble antioxidants.22 23 The incubation of LDL with millimolar concentrations of ONOO- results in lipoprotein aggregation and conversion into a high uptake form for macrophages.7 24 In the presence of CO2, ONOO- forms a more potent nitrating intermediate, presumably nitrosoperoxocarbonate (ONOOCO2-).25 At physiological concentrations of CO2, the yield of ONOO--mediated nitration reactions is increased, apparently through formation of this adduct (reviewed in Reference 2626 ). The formation of ONOO- is shown in the following equation:

(1)

Two alternative pathways for generating reactive nitrogen species were recently described; they each involve MPO, a protein secreted by neutrophils and monocytes.27 28 29 30 Immunohistochemical studies demonstrate that MPO is abundant in human atherosclerotic lesions.31 LDL recovered from human atheromas is enriched in chlorotyrosine, a specific product of MPO-generated hypochlorous acid (HOCl).32 Antibodies specific for nitrotyrosine recognize epitopes in human atheroma that colocalize with mononuclear phagocytic cells,5 which are similar to MPO.31 Recent studies demonstrate that isolated MPO can use nitrite (NO2-), a major end product of •NO metabolism,11 33 as a substrate to nitrate protein tyrosine residues7 28 29 and initiate lipid peroxidation.7 34

LDL exposed to the MPO-H2O2-NO2- system is converted into a high-uptake form for macrophages, leading to cholesterol deposition and foam cell formation,7 essential steps in atherogenesis. The reactive nitrogen species formed during MPO-catalyzed oxidation of NO2- have not yet been identified, but nitrogen dioxide (•NO2), the one-electron oxidation product of NO2-, has been implicated.28 Finally, MPO may also indirectly generate a nitrating intermediate through the secondary oxidation of NO2- with HOCl, presumably forming nitryl chloride (NO2Cl).27 Exposure of LDL to NO2Cl causes lipoprotein aggregation and conversion into a phagocytosable particle for macrophages.35 Despite the ability of monocytes to undergo a respiratory burst, to generate •NO, and to secrete MPO when activated, direct evidence that monocytes can generate reactive nitrogen species that promote LDL nitration and lipid peroxidation is lacking. Equations for the chemical reactions discussed above follow:

(2)

(3)

In the present study, we examined the various chemical pathways available to monocytes for generating reactive nitrogen species. Mechanisms for the monocyte-dependent formation of nitrating intermediates and characterization of the ability of these distinct pathways to promote LDL protein nitration and initiate LDL lipid peroxidation are presented. Finally, we examined the role of •NO production rates on the relative contribution of distinct pathways in activated monocytes for promoting apo B-100 nitration and LDL lipid peroxidation.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
All media were purchased from Life Technologies. E,Z-9-hydroxy-10,12-octadecadienoic acid (9-HODE), 8-epi-prostaglandin F2{alpha} (PGF2{alpha}), and 8-epi-[2H4]PGF2{alpha} standards were purchased from Cayman Chemical, and [3H]arachidonate (40 mCi/mmol) was obtained from ICN Pharmaceutical, Inc. Organic solvents (high-performance liquid chromatography [HPLC]–grade) were obtained from Fisher Chemical Company. Catalase (bovine liver, thymol-free) and glucose oxidase (grade II) were purchased from Boehringer Mannheim. All other reagents were obtained from Sigma Chemical Co, unless otherwise specified.

Methods
General Procedures
All buffers and media were treated with Chelex-100 resin (Bio-Rad) and supplemented with diethylenetriaminepentaacetic acid (DTPA) to remove trace levels of potential redox-active transition metal ions. Protein content was determined by the Markwell-modified Lowry protein assay36 using bovine serum albumin as the standard. The concentration of reagent H2O2 was determined spectrophotometrically ({epsilon}240=39.4 mol-1 · L · cm-1; Reference 3737 ). NO2-/NO3- production by human monocytes was monitored by the Griess assay.38

Oxidation Reactions
Human monocytes were isolated from the peripheral blood of healthy volunteers after obtaining informed consent. All protocols were approved by the Cleveland Clinic Foundation Institutional Review Committee and were in accordance with institutional guidelines. Preliminary studies demonstrated that monocytes isolated by methods that use adhesion/washing steps contained significantly less MPO per cell (presumably because of premature degranulation during the isolation procedure). Human peripheral blood monocytes used for this study were therefore isolated by elutriation.39 Monocytes were incubated at 37°C under 95% air and 5% CO2 in medium A (Ca2+/Mg2+/phenol-free Hanks’ balanced salt solution [Gibco Life Technologies] supplemented with 50 µmol/L L-arginine and 200 µmol/L DTPA [pH 7.4 final]) containing 0.2 mg/mL LDL protein in the presence or absence of NaNO2 (0 to 50 µmol/L, as indicated in the figure legends).

Monocytes (1x106 cells/mL) were activated with 200 nmol/L phorbol myristate acetate (PMA) and maintained in suspension by gentle mixing every 5 minutes. After 2 hours, reactions were stopped by creating cell pellets (by centrifugation [2000g] at 4°C) and adding 50 µmol/L butylated hydroxytoluene to the supernatants. LDL oxidation products were then assayed in supernatants as described below. In some experiments, monocytes were activated in the presence of an exogenous •NO source by adding Z-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA NONOate, Alexis Corp) 1 minute after the addition of PMA. Initial concentrations of the NO donor were selected on the basis of calculated rates of •NO flux, assuming that PAPA NONOate spontaneously releases 2 mol of •NO per mol of donor molecule, with a t1/2 of 15 minutes at 37°C (pH 7.4).40 Actual rates of NO flux were then determined spectrophotometrically by the reaction of NO with oxyhemoglobin41 under the same conditions used for experiments but in the absence of any added cells. To maintain a final pH of 7.4 during experiments with PAPA NONOate, incubations were performed in medium B (medium A containing only 100 mmol/L NaCl and supplemented with 20 mmol/L sodium phosphate).

Pilot experiments revealed that detectable levels of F2-isoprostanes were generated by monocytes in the absence of added NO2-, but only at a level {approx}20% of that observed in the presence of NO2-. This was presumably due to prostaglandin endoperoxide synthase activity, because the reaction was inhibited by aspirin and dexamethasone and not inhibited by superoxide dismutase (SOD), aminotriazole, or butylated hydroxytoluene. Thereafter, in all experiments in which F2-isoprostane production was measured, the contribution of prostaglandin endoperoxide synthase-1 and -2 to eicosanoid production was suppressed by including aspirin (10 µg sodium salicylate/mL) and dexamethasone (2 µmol/L), respectively, in reaction mixtures before monocyte stimulation.42

Sample Preparation and Mass Spectrometric Analyses
Nitrotyrosine content was determined by stable isotope dilution gas chromatography–mass spectrometry (GC/MS) on a Finnigin Voyager quadrupole GC/MS equipped with a chemical ionization probe. 3-[13C6]nitrotyrosine was synthesized and used as an internal standard, as previously described.6 Briefly, reactions were terminated by forming cell pellets and adding butylated hydroxytoluene. Lipids and salts were removed by extraction with a single-phase solvent mixture of H2O:water-washed diethyl ether:methanol (1:3:7, vol:vol:vol).43 Internal standards were then added, samples were hydrolyzed with HCl, and the nitrotyrosine content in amino acid hydrolysates was determined after reduction to amino tyrosine as an n-propyl, per heptafluorobutyryl derivative.44 Nitrotyrosine content was normalized to the content of tyrosine determined by stable isotope dilution GC/MS45 using [2H4]-labeled tyrosine as the standard. Under the conditions used, no significant intrapreparative formation of nitrotyrosine, which was routinely assessed in each sample by determining the 3-[2H3]-nitrotyrosine present, occurred.

For 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadienoate acid (9-H[P]ODE) and F2-isoprostane determinations, hydroperoxides in reaction mixtures were reduced to their corresponding stable hydroxides during extraction using a modified Dole procedure in which the reducing agent, triphenylphosphine, was present.46 These conditions also inhibited artifactual formation of F2-isoprostanes and oxidized lipids.47 Lipids were dried under N2 and resuspended in isopropanol (2 mL); then, fatty acids were released by base hydrolysis with 1N sodium hydroxide (2 mL) at room temperature under N2 for 90 minutes. The samples were acidified (pH 3.0) with 2N HCl, known amounts of internal standards were added, and free fatty acids were extracted twice with hexane (5 mL). The content of 9-H(P)ODEs and F2-isoprostanes were then determined by HPLC with online tandem mass spectrometric (LC/MS/MS) analysis. Control experiments using [3H]arachidonate confirmed that no detectable formation of [3H]-labeled 9-H(P)ODE or F2-isoprostanes occurred under the conditions used for sample preparation.

LC/MS/MS analyses were performed on a Quatro II triple quadruple mass spectrometer (Micromass, Inc) interfaced with an HP 1100 HPLC (Hewlett Packard). F2-isoprostanes were quantified by stable isotope dilution mass spectrometry using online reverse-phase LC/MS/MS with 8-epi-[2H4]PGF2{alpha} as the standard.48 For 9-HODE analyses, lipid extracts generated after base hydrolysis of reduced lipids (above) were dried under N2 and reconstituted in 100 µL of methanol. An aliquot of the mixture was then injected on an Ultrasphere ODS C18 column (4.6x250 mm, 5 µm, Beckman Instruments), equilibrated, and run under isocratic conditions at 1 mL/min using methanol:H2O, (85:15, vol/vol) as the solvent. The column eluent was split (930 µL/min to UV detector and 70 µL/min to mass detector) and analyzed by the mass spectrometer. The LC/MS/MS analyses of 9-HODE and F2-isoprostanes in column effluents were performed using electrospray-ionization mass spectrometry in the negative-ion mode with multiple-reaction monitoring and monitoring the transitions at mass per unit charge (m/z) 295->171 for 9-HODE, m/z 353->309 for F2-isoprostanes, and m/z 357->313 for [2H4]PGF2{alpha}. The endogenous content of 9-H(P)ODE in samples was initially determined by the method of standard additions using known amounts of authentic 9-HODE added to samples. Quantification in subsequent experiments was done using external calibration curves constructed with authentic 9-HODE after preliminary LC/MS/MS experiments demonstrating results identical to those obtained by the method of standard additions.

Statistics
Statistical analyses were done using a paired Student’s t test. For all hypotheses, the significance level was 0.05. When multiple comparisons were made, a Bonferroni correction to the significance criterion for each test was done.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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LDL Protein Nitration and Lipid Peroxidation Are Mediated by the MPO-H2O2-NO2- System of Monocytes
Fresh peripheral blood monocytes were activated with PMA in Chelex-100–treated media containing the metal chelator DTPA and LDL, and the extent of LDL modification by reactive nitrogen species was determined by stable isotope dilution GC/MS analysis of apo B-100 nitrotyrosine content. In parallel, the ability of monocyte-generated reactive nitrogen species to initiate lipid peroxidation was assessed by quantifying the oxidation products of linoleic acid (9-H(P)ODE) and arachidonic acid (F2-isoprostanes) by LC/MS/MS. Under these conditions, monocyte activation failed to promote protein nitration or lipid peroxidation of LDL. In contrast, adding NO2- to the monocyte reaction mixtures at concentrations comparable to those observed in inflammatory tissues and fluids (See Reference 2828 and the references therein) induced the nitration of apo B-100 tyrosine residues and initiated LDL lipid peroxidation (Figure 1Down). The time course for tyrosine nitration and lipid peroxidation paralleled the time course for O2•- production and was maximal by {approx}120 minutes (data not shown). Tyrosine nitration and lipid peroxidation by monocytes required cell activation and were inhibited by the presence of peroxidase inhibitors (NaN3 and aminotriazole). Moreover, adding catalase, but not heat-inactivated catalase, effectively blocked nitrotyrosine, 9-H(P)ODE, and F2-isoprostane production (Figure 1Down).



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  		Figure 1. Monocytes activated in media containing NO2--nitrate apo B-100 tyrosine residues and initiate lipid peroxidation via the MPO/H2O2 system. Isolated human monocytes (1x106 cells/mL) were incubated at 37°C in medium A in presence of LDL (0.2 mg/mL) and NO2- (50 µmol/L). Monocytes (Mono) were activated with 200 nmol/L PMA and maintained in suspension by intermittent inversion (Complete System) for 2 hours. Additions or deletions to the Complete System were as indicated. Reactions were stopped by removing cells through centrifugation. Left, Content of nitrotyrosine (NY) in LDL was then determined by stable-isotope dilution GC/MS, as described in Materials and Methods. Middle, Lipids in supernatants were extracted, reduced, and hydrolyzed, and content of total (free+lipid-bound) 9-H(P)ODE was determined. Right, F2-isoprostanes were then determined by LC/MS/MS, as described in Materials and Methods. Final concentrations of additions to Complete System were 1 mmol/L NaN3, 10 mmol/L 3-aminotriazole (Atz), 300 nmol/L catalase (Cat), 300 nmol/L heat-inactivated catalase (hiCat), 100 µmol/L methionine (Met), 10 µg/mL SOD, and 10 µg/mL heat-inactivated SOD (hiSOD). Data represent mean±SD of triplicate determinations. *P<0.05 for comparison vs Complete System. Y indicates tyrosine.

Monocytes activated with N-formyl-methionyl-leucyl-phenylalanine (100 nmol/L) similarly demonstrated NO2--dependent LDL tyrosine nitration and lipid peroxidation, albeit at a level {approx}6- to 8-fold less (2 fmol O2•-/cell over a 2-hour interval) than that observed with monocytes stimulated with PMA (12 fmol O2•-/cell over a 2-hour interval), which is consistent with the findings that both O2•- production and MPO secretion are decreased in monocytes stimulated with this agonist. Finally, the addition of methionine, a potent scavenger of chlorinating oxidants like HOCl and NO2Cl, failed to significantly attenuate the extent of tyrosine nitration and lipid peroxidation induced by the activated monocytes (Figure 1Up). These results suggest that secondary oxidation of NO2- by HOCl (eg, Equation 3Up) was not a significant pathway for reactive nitrogen species formation by the activated monocytes.

Interestingly, adding either SOD or heat-inactivated SOD to the reaction mixture did not attenuate protein nitration or lipid peroxidation by the cells, suggesting that O2•- (and hence ONOO-; Equation 1Up) does not play a direct role in LDL oxidation by human peripheral blood monocytes under the conditions examined. Rather, adding SOD to the activated cells resulted in a modest increase ({approx}50%) in the production of markers of lipid peroxidation (Figure 1Up). Control experiments suggested that SOD itself was not directly promoting lipid peroxidation because the addition of both SOD and peroxidase inhibitors (ie, NaN3, NaCN, or aminotriazole) to cells activated in the presence of NO2- resulted in no significant formation of lipid oxidation products (data not shown). The mechanism(s) responsible for the increased extent of lipid peroxidation observed in the presence of SOD are unknown, but they may reflect the prevention of O2•--dependent conversion of MPO into an inactive form (ie, Compound III)49 or the enhanced conversion of superoxide (O2•-) into the MPO substrate H2O2.50 Alternatively, increased levels of lipid peroxidation with the addition of SOD could result from excess scavenging of the O2•- radical, preventing it from terminating chain-propagating reactions and leading to a net increase in lipid peroxidation via increased chain length.51 Collectively, these results suggest that freshly isolated human peripheral blood monocytes generate reactive nitrogen species primarily through a peroxidase-, H2O2- and NO2--dependent pathway (ie, the MPO-H2O2-NO2- pathway; Equation 2Up).

Monocytes Use Physiologically Relevant Levels of NO2- to Nitrate Apo B-100 Tyrosine Residues and Initiate LDL Lipid Peroxidation
To further examine the physiological relevance of NO2- oxidation by monocytes as a potential mechanism for the modification of protein and lipid targets in vivo, we activated cells in the presence of LDL and concentrations of NO2- similar to those observed in vivo (ie, <=50 µmol/L; reviewed in Reference 2828 ) and then monitored the extent of tyrosine nitration and lipid peroxidation by stable isotope dilution GC/MS and LC/MS/MS, respectively. In the presence of normal plasma levels of NO2- (ie, <=5 µmol/L), monocyte activation resulted in LDL nitration and lipid peroxidation. Moreover, nitration of apo B-100 tyrosine residues (Figure 2Down) and peroxidation of LDL lipids (Figure 3Down) increased progressively as a function of NO2- concentration over a pathophysiologically relevant range. Adding catalase to the reaction mixtures before cell activation inhibited protein and lipid modification by the reactive nitrogen intermediate(s) formed during monocyte-dependent oxidation of NO2-. Thus, monocyte activation in vivo will likely produce nitrating intermediates at both normal and pathophysiologically relevant concentrations of NO2- through the MPO-H2O2-NO2- system.



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  		Figure 2. NO2- concentration-dependence of monocyte-mediated nitration of apo B-100 tyrosine residues. LDL (0.2 mg/mL) was incubated with phorbol ester (200 nmol/L) stimulated human monocytes (106 cells/mL) and indicated concentrations of NO2- in absence (•) and presence ({circ}) of 300 nmol/L catalase (Cat) in medium A for 2 hours at 37°C. Reactions were stopped by removing cells through centrifugation, and content of nitrotyrosine in LDL was then determined by stable isotope dilution GC/MS, as described in Materials and Methods. Data represent mean±SD of triplicate determinations.



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  		Figure 3. NO2- concentration-dependence of monocyte-mediated initiation of LDL lipid peroxidation. LDL (0.2 mg/mL) was incubated with phorbol ester (200 nmol/L) stimulated human monocytes (106 cells/mL) and indicated concentrations of NO2- in absence (•) and presence ({circ}) of 300 nmol/L catalase (Cat) in medium A for 2 hours at 37°C. Reactions were stopped by removing cells through centrifugation. Lipids in supernatants were extracted, reduced, and hydrolyzed. Then, content of (top) total (free+lipid-bound) 9-H(P)ODE and (bottom) F2-isoprostanes were determined by LC/MS/MS, as described in Materials and Methods. Data represent mean±SD of triplicate determinations.

Monocytes Generate •NO-Derived Oxidants
One striking feature of the results described thus far was that we could find no evidence that isolated monocytes generate nitrating intermediates through the formation of ONOO- (Equation 1Up), because both nitrotyrosine formation and lipid peroxidation required the addition of NO2- to the media (Figures 1 through 3UpUpUp). This likely reflects the limited capacity of freshly isolated peripheral blood monocytes to generate •NO, the brief time period used for LDL incubation with monocytes (ie, 2 hours, the approximate duration of O2•- formation after a PMA-stimulated respiratory burst), and the presence of metal chelators in cell culture media to prevent lipid peroxidation mediated by trace levels of redox-active transition metal ions. Indeed, we were unable to detect any endogenous rate of NO production (or NO2-/NO3- accumulation) in human monocytes freshly isolated from peripheral blood, with or without phorbol ester activation, under the conditions and time course (2 hours) used. To more fully explore the chemical mechanisms available to monocytes for generating reactive nitrogen species, we performed a series of experiments in which monocytes were activated in the presence of an exogenous •NO-generating system, PAPA NONOate.40 These conditions were expected to more closely mimic a physiological mechanism for NO2- formation and to provide an environment in which monocyte-generated O2•- might react with •NO before it dismutates into the MPO substrate H2O2.

Although monocytes activated in the absence of the •NO donor failed to nitrate apo B-100 tyrosine residues or to produce detectable levels of the products of lipid peroxidation (ie, 9H(P)ODE and F2-isoprostanes), cells stimulated in the presence of a continuous •NO source readily promoted LDL protein (Figure 4Down) and lipid (Figure 5Down) modification. Protein nitration and lipid peroxidation by monocytes required cell activation, which is consistent with a requirement for reduced oxygen species (O2•- and/or H2O2) for lipoprotein oxidation (data not shown). At higher fluxes of •NO, the overall extent of nitrotyrosine formation and lipid peroxidation diminished (Figures 4Down and 5Down). Taken together, the present results demonstrate that activated monocytes generate •NO-derived oxidants that can damage protein and lipid targets.



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  		Figure 4. Nitration of apo B-100 tyrosine residues by monocyte-generated •NO-derived oxidants. LDL (0.2 mg/mL) was incubated for 2 hours at 37°C with phorbol ester (200 nmol/L) stimulated human monocytes (106 cells/mL) in presence of increasing rates of •NO release from PAPA NONOate, as described in Materials and Methods. Reactions were stopped by removing cells through centrifugation. Content of nitrotyrosine formed in LDL was then determined by stable-isotope dilution GC/MS, as described in Materials and Methods. Data represent mean±SD of triplicate determinations.



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  		Figure 5. Initiation of LDL lipid peroxidation by monocyte-generated •NO-derived oxidants. LDL (0.2 mg/mL) was incubated for 2 hours at 37°C with phorbol ester (200 nmol/L) stimulated human monocytes (106 cells/mL) in presence of increasing rates of •NO release from PAPA NONOate, as described in Materials and Methods. Reactions were stopped by removing cells through centrifugation. Lipids in supernatants were extracted, reduced, and hydrolyzed, and content of total (free+ lipid-bound) 9-H(P)ODE formed was then determined by LC/MS/MS, as described in Materials and Methods. Data represent mean±SD of triplicate determinations.

Monocytes Use Distinct Pathways for Generating •NO-Derived Oxidants
At low rates of •NO flux, LDL protein nitration (Figure 6Down, top) and lipid peroxidation (Figure 7Down, top) were inhibited by either catalase or azide. In contrast, under these conditions, the addition of either HOCl scavengers (eg, methionine or N{alpha}-acetyl lysine) or SOD failed to significantly attenuate nitrotyrosine or 9-H(P)ODE production (Figure 6Down, bottom, and Figure 7Down, bottom, respectively). These results suggest that at low rates of •NO flux, LDL modification by monocytes occurs through a pathway requiring H2O2 and MPO (ie, the MPO-H2O2-NO2- system; Equation 2Up). They also suggest that monocyte-mediated nitration and lipid peroxidation reactions at low levels of •NO flux do not proceed through the intermediary formation of HOCl or ONOO- (ie, Equations 1Up, and 3Up).



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  		Figure 6. Effect of MPO-inhibitors, catalase, SOD, and HOCl scavengers on nitration of apo B-100 tyrosine residues by monocyte-generated •NO-derived oxidants. LDL (0.2 mg/mL) was incubated for 2 hours at 37°C with phorbol ester (200 nmol/L) stimulated human monocytes (106 cells/mL) in presence of increasing rates of •NO release from PAPA NONOate, as described in Figure 4Up (control condition). In parallel reactions, cells were also incubated with indicated addition. Reactions were stopped by removing cells through centrifugation, and content of nitrotyrosine formed in LDL was then determined by stable-isotope dilution GC/MS, as described in Materials and Methods. Data represent mean±SD of triplicate determinations and are expressed as percent of nitrotyrosine formed in presence of indicated addition relative to level of nitrotyrosine formed under control conditions. Similar results were observed in 3 independent experiments. Concentration of additives, when present, were as follows: 300 nmol/L catalase (Cat), 1 mmol/L NaN3, 1 mmol/L N{alpha}-acetyl lysine (N{alpha}-AcLys), 1 mmol/L methionine (Met), and 10 µg/mL SOD.



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  		Figure 7. Effect of MPO-inhibitors, catalase, SOD, and HOCl scavengers on lipid peroxidation initiated by monocyte-generated •NO-derived oxidants. LDL (0.2 mg/mL) was incubated for 2 hours at 37°C with phorbol ester (200 nmol/L) stimulated human monocytes (106 cells/mL) in presence of increasing rates of •NO release from PAPA NONOate, as described in Figure 5Up (control condition). In parallel reactions, cells were also incubated with indicated addition. Reactions were stopped by removing cells through centrifugation. Lipids in supernatants were extracted, reduced, and hydrolyzed, and content of total (free+lipid-bound) 9-H(P)ODE formed was then determined by LC/MS/MS, as described in Materials and Methods. Data represent mean±SD of triplicate determinations and are expressed as percent of total 9-H(P)ODE formed in presence of indicated addition relative to level formed under control conditions. Concentration of additives, when present, were as follows: 300 nmol/L catalase (Cat), 1 mmol/L NaN3, 1 mmol/L N{alpha}-acetyl lysine (N{alpha}-AcLys), 1 mmol/L methionine (Met), and 10 µg/mL SOD.

At high rates of •NO flux, LDL protein nitration (Figure 6Up, top) and lipid peroxidation (Figure 7Up, top) by activated monocytes became increasingly less sensitive to inhibition by either catalase or azide. These results suggest that the relative contribution of the MPO-H2O2-NO2- system toward the generation of reactive nitrogen species by monocytes diminishes under these conditions. Although adding methionine partially attenuated the extent of nitrotyrosine and 9-H(P)ODE produced at high levels of •NO flux, the mechanism does not seem to be due to the consumption of NO2Cl or HOCl. Adding a primary amine (N{alpha}-acetyl lysine) that also reacts rapidly with reactive chlorinating species like HOCl and NO2Cl to form monochloramines,52 53 agents which do not promote the secondary oxidation of NO2-,29 failed to inhibit either nitrotyrosine production or LDL lipid peroxidation by activated monocytes (Figure 6Up, bottom, and Figure 7Up, bottom, respectively). Taken together, the present results indicate that monocytes use distinct pathways for generating •NO-derived oxidants.


*    Discussion
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 *Discussion
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Reactive nitrogen species are implicated in many inflammatory and vascular disorders. Because of the ability of monocytes to generate both •NO and O2•- and the central role monocytes seem to play in atherogenesis, they are generally assumed to represent a cellular source of •NO-derived oxidants such as ONOO- in vivo. However, the chemical pathways available to monocytes for generating •NO-derived oxidants have not yet been directly examined. Indeed, neither the cellular sources of nitrating intermediates nor the reaction pathways for their generation in vivo are established.17

The present studies demonstrated that monocytes use distinct mechanisms for generating reactive nitrogen intermediates. They also demonstrated that the environment in which monocyte activation occurs (eg, level of •NO) influences the relative contribution that distinct oxidation pathways play in contributing to protein nitration and lipid peroxidation. On the basis of the results from the present report and prior published studies, we generated the following model (Figure 8Down) of pathways available to monocytes for promoting LDL modification by reactive nitrogen species. On activation, the NADPH oxidase complex of monocytes forms O2•-, which both spontaneously and enzymatically (through SOD) dismutates to form H2O2.13 Concomitantly, monocyte activation leads to the secretion of MPO into the extracellular compartment (and into the phagolysosomal compartment; not shown).53 Under conditions of relatively low levels of •NO flux, the primary pathway used by monocytes for generating reactive nitrogen intermediates is through MPO/H2O2-catalyzed oxidation of NO2- (Figure 8Down, pathway A). The ability of monocytes to use this pathway is strongly supported by the demonstration that both activated monocytes (this study) and isolated MPO7 use physiological concentrations of NO2- to nitrate apo B-100 tyrosine residues and initiate LDL lipid peroxidation. The ability of MPO inhibitors (NaN3, NaCN, and aminotriazole) and H2O2 scavengers (catalase) to block nitrotyrosine (Figures 1Up and 6Up), F2-isoprostane (Figure 1Up), and 9-H(P)ODE (Figures 1Up and 7Up) formation by cells activated in the presence of either NO2- or •NO are consistent with a primary role for the MPO-H2O2-NO2- pathway in the formation of nitrating oxidants by monocytes. The reactive nitrogen species (NOx) formed by the MPO-catalyzed oxidation of NO2- has not yet been identified. Both one-electron (nitrogen dioxide)11 28 and two-electron (NO2+ or ONOO-)11 54 oxidation products have been suggested as potential nitrating intermediates formed during the peroxidase-catalyzed oxidation of NO2-. Further studies to identify the nitrating intermediate(s) produced during MPO-catalyzed oxidation of NO2- are warranted.



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  		Figure 8. Model of potential pathways available to monocytes for generation of •NO-derived oxidants.

The present study also confirms that another pathway for monocytes to generate reactive nitrogen species is through the formation of ONOO- (Figure 8Up, pathway C). Although monocytes from freshly isolated human peripheral blood failed to generate detectable levels of nitrotyrosine or lipid oxidation products in LDL exposed to activated cells only, monocyte stimulation in the presence of high fluxes of •NO resulted in LDL nitration and lipid peroxidation. The ability of SOD and methionine (which scavenge O2•- and ONOO-, respectively) to inhibit nitrotyrosine and 9-H(P)ODE production (Figures 6Up and 7Up, bottom) and the lack of significant inhibition in the presence of MPO inhibitors or catalase (Figures 6Up and 7Up, top) support a role for ONOO- (Figure 8Up, pathway C), and not the MPO-H2O2-NO2- system (Figure 8Up, pathway A), in promoting LDL modification under these conditions. Thus, under conditions of high •NO flux, O2•- interaction with •NO forming ONOO- (ie, Equation 1Up) seems to account for a significant portion of the reactive nitrogen species formed by activated monocytes.

It is interesting to note that the overall yields of nitration and lipid peroxidation dropped at higher fluxes of •NO (Figures 4Up and 5Up). Several mechanism(s) may account for these results. One likely reason is that •NO may partially act as an antioxidant under these conditions by scavenging reactive intermediates though radical-radical interactions.55 Alternatively, the decreased formation of nitrotyrosine and lipid oxidation products may be due to lower overall yields with ONOO- compared with the MPO-H2O2-NO2- system. Another intriguing possibility is that high levels of •NO flux could potentially attenuate protein nitration and lipid peroxidation by inhibiting critical heme proteins involved in O2•- formation (eg, NADPH oxidase) or reactive nitrogen species formation (eg, MPO) (H.M. Abu-Soud and S.L. Hazen, unpublished data, 1999).

We were unable to detect any endogenous rate of •NO production (or NO2-/NO3- accumulation) in human monocytes freshly isolated from peripheral blood, with or without phorbol ester activation, under the conditions and time course used. Similarly, replacement of L-arginine in the media with L-nitro-arginine methyl ester, an inhibitor of NOS, had no effect on the level of oxidation products generated by activated monocytes in the presence or absence of exogenous NO2- or •NO (data not shown). These results are consistent with the low levels of the inducible isoform of NOS found in freshly isolated human monocytes; however, in the presence of a variety of cytokines and vasoactive hormones, human monocytes can generate •NO after the upregulation of NOS gene expression.12 Thus, both monocytes and endothelium may serve as •NO sources in vivo and contribute to ONOO- (and NO2-) formation.

Although the secondary oxidation of NO2- by MPO-generated HOCl is reported as a mechanism for MPO- and neutrophil-mediated nitrotyrosine formation,27 29 the present results failed to demonstrate a role for this pathway (Figure 8Up, pathway B) in the monocyte-dependent formation of nitrating agents. Specifically, neither the extent of apo B-100 nitrotyrosine formation nor the content of F2-isoprostanes and 9-H(P)ODE formed during exposure of LDL to activated monocytes and NO2- was blocked by the addition of the HOCl scavenger methionine (Figure 1Up). Moreover, monocyte activation in the presence of an •NO donor and N{alpha}-acetyl lysine, which rapidly reacts with HOCl and NO2Cl to form monochloramines, failed to inhibit LDL nitration and lipid peroxidation (Figures 6Up and 7Up, bottom). Whether this pathway contributes to reactive nitrogen species formation by monocytes activated under alternative conditions remains unknown.

An important consideration is the concentrations of NO2-, •NO, and CO2-HCO3- used in the present study. Although the actual levels of NO2- present in tissues such as the arterial intima are not known, NO2- levels of <=5 µmol/L are normally present in plasma, and levels as high as 50 µmol/L have been observed in numerous biological fluids during inflammation (See Reference 2828 and references therein). Thus, the present results demonstrate that monocytes use biologically relevant levels of NO2- to promote protein nitration and lipid peroxidation (Figures 2Up and 3Up). Similarly, a wide range of steady-state levels of •NO are observed (20 nmol/L to 2 µmol/L) in vivo.11 •NO-dependent protein nitration and lipid peroxidation were mediated by monocytes activated in the presence of •NO fluxes over this range (Figures 4Up and 5Up). Finally, recent studies demonstrated that the chemical reactivity and actions of ONOO- are significantly influenced by CO2-HCO3- (reviewed in Reference 2626 ). Intra- and extracellular concentrations of CO2 and HCO3- in humans are typically 1 to 2 mmol/L and 7 to 25 mmol/L, respectively.56 At these levels, a significant portion of the reactivity of ONOO- in solution is attributable to ONOOCO2-.25 26 27 57 Although the levels of CO2 (5% gas phase) and HCO2- in the media (4.2 mmol/L) used in these studies more closely mimic a biologically relevant situation, the proximate oxidant in reactions mediated by the product of •NO and O2•- (Figure 8Up, pathway C) is not easily established (ONOO- versus ONOOCO2- or a combination of the two).

A significant finding of the present study is the demonstration that alternative pathways to ONOO- formation can play a significant—and even predominant—role in oxidative injury of biomolecules by monocyte-generated nitrating intermediates. One practical consequence is that efforts to block oxidative injury in vivo by •NO-derived oxidants will require the use of distinct pharmacological agents that block distinct pathways. For example, agents such as SOD mimetics, which can compete with •NO for O2•- and inhibit ONOO- formation, may serve to paradoxically enhance nitration and lipid peroxidation reactions through the MPO-H2O2-NO2- pathway. It is known that nitrotyrosine is present in a variety of tissues and conditions. What remains is to determine the functional consequences of protein and lipid modification by nitrating oxidants in vivo and to identify which factors influence the relative contributions of various mechanisms for generating •NO-derived oxidants during human health and disease.


*    Acknowledgments
 
This work was supported in part by a grant from the American Heart Association and by grants HL62526, HL61878, and HL53315 from the National Institutes of Health. Mass spectrometry experiments were performed at the Cleveland Mass Spectrometry Core Facility.

Received June 17, 1999; accepted September 8, 1999.


*    References
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 *References
 

  1. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785–1792.
  2. Steinbrecher UP, Zhang HF, Lougheed M. Role of oxidatively modified LDL in atherosclerosis. Free Radic Biol Med. 1990;9:155–168.[Medline] [Order article via Infotrieve]
  3. Chisolm GM, Penn MS. Oxidized lipoproteins and atherosclerosis. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. Philadelphia: Lippincott-Raven; 1998:129–149.
  4. Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA, Watson AD, Lusis AJ. Atherosclerosis: basic mechanisms, oxidation, inflammation, and genetics. Circulation. 1995;91:2488–2496.[Abstract/Free Full Text]
  5. Beckman JS, Ye YZ, Anderson PG, Chen J, Accavitti MA, Tarpey MM, White R. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe Seyler. 1993;375:81–88.
  6. Leeuwenburgh C, Hardy MM, Hazen SL, Wagner P, Ohishi S, Steinbrecher UP, Heinecke JW. Reactive nitrogen intermediates promote low density lipoprotein oxidation in human atherosclerotic intima. J Biol Chem. 1997;272:1433–1436.[Abstract/Free Full Text]
  7. Podrez EA, Schmitt D, Hoff HF, Hazen SL. Myeloperoxidase-generated reactive nitrogen species convert LDL into an atherogenic form in vitro. J Clin Invest. 1999;103:1547–1563.[Medline] [Order article via Infotrieve]
  8. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993;329:2002–2012.[Free Full Text]
  9. Beckman JS, Chen J, Ischiropoulos H, Crow JP. Oxidative chemistry of peroxynitrite. Methods Enzymol. 1994;233:229–240.[Medline] [Order article via Infotrieve]
  10. van der Vliet A, Eiserich JP, Kaur H, Cross CE, Halliwell B. Nitrotyrosine as biomarker for reactive nitrogen species. Methods Enzymol. 1996;269:175–184.[Medline] [Order article via Infotrieve]
  11. Patel RP, McAndrew J, Sellak H, White CR, Jo H, Freeman BA, Darley-Usmar VM. Biological aspects of reactive nitrogen species. Biochem Biophys Acta. 1999;1411:385–400.[Medline] [Order article via Infotrieve]
  12. Forstermann U, Kleinert H, Gath I, Schwartz P, Closs EI, Dun NJ. Expression and expressional control of nitric oxide synthases in various cell types. In: Ignarro L, Murad F, eds. Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications. San Diego, Calif: Academic Press; 1995:171–186.
  13. Babior BM. Oxygen-dependent microbial killing by phagocytes. N Engl J Med. 1978;298:659–663.[Medline] [Order article via Infotrieve]
  14. Klebanoff SJ. Oxygen metabolism and the toxic properties of phagocytes. Ann Intern Med. 1980;93:480–489.
  15. Smith JD, Trogan E, Ginsberg M, Grigaux C, Tian J, Miyata M. Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E. Proc Natl Acad Sci U S A. 1995;92:8264–8268.[Abstract/Free Full Text]
  16. Jessup W, Mohr D, Gieseg SP, Dean RT, Stocker R. The participation of nitric oxide in cell free- and its restriction of macrophage-mediated oxidation of low-density lipoprotein. Biochem Biophys Acta. 1992;1180:73–82.[Medline] [Order article via Infotrieve]
  17. Halliwell B. What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo? FEBS Lett. 1997;411:157–160.[Medline] [Order article via Infotrieve]
  18. Hogg N, Struck A, Goss SP, Santanam N, Joseph J, Parthasarathy S, Kalyanaraman B. Inhibition of macrophage-dependent low density lipoprotein oxidation by nitric-oxide donors. J Lipid Res. 1995;36:1756–1762.[Abstract]
  19. Goss SP, Hogg N, Kalyanaraman B. The effect of nitric oxide release rates on the oxidation of human low density lipoprotein. J Biol Chem. 1997;272:21647–21653.[Abstract/Free Full Text]
  20. Darley-Usmar VM, Hogg N, O’Leary VJ, Wilson MT, Moncada S. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Radic Res Commun. 1992;17:9–20.[Medline] [Order article via Infotrieve]
  21. Radi R, Beckman JS, Bush KM, Freeman BA. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys. 1991;288:481–487.[Medline] [Order article via Infotrieve]
  22. Hogg N, Darley-Usmar VM, Wilson MT, Moncada S. The oxidation of {alpha}-tocopherol in human low-density lipoprotein by the simultaneous generation of superoxide and nitric oxide. FEBS Lett. 1993;326:199–203.[Medline] [Order article via Infotrieve]
  23. Goss SP, Hogg N, Kalyanaraman B. The effect of {alpha}-tocopherol on the nitration of {gamma}-tocopherol by peroxynitrite. Arch Biochem Biophys. 1999;363:333–340.[Medline] [Order article via Infotrieve]
  24. Graham A, Hogg N, Kalyanaraman B, O’Leary V, Darley-Usman V, Moncada S. Peroxynitrite modification of low-density lipoprotein leads to recognition by the macrophage scavenger receptor. FEBS Lett. 1993;330:181–185.[Medline] [Order article via Infotrieve]
  25. Lymar SV, Jiang Q, Hurst JK. Mechanism of carbon dioxide-catalyzed oxidation of tyrosine by peroxynitrite. Biochemistry. 1996;35:7855–7861.[Medline] [Order article via Infotrieve]
  26. Radi R, Denicola A, Freeman BA. Peroxynitrite reactions with carbon dioxide-bicarbonate. Methods Enzymol. 1999;301:353–367.[Medline] [Order article via Infotrieve]
  27. Eiserich JP, Cross CE, Jones AD, Halliwell B, van der Vleit A. Formation of nitrating and chlorinating species by reaction of nitrite with hypochlorous acid: a novel mechanism for nitric oxide-mediated protein modification. J Biol Chem. 1996;271:19199–19208.[Abstract/Free Full Text]
  28. van der Vleit A, Eiserich JP, Halliwell B, Cross CE. Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite: a potential additional mechanism of nitric oxide-dependent toxicity. J Biol Chem. 1997;272:7617–7625.[Abstract/Free Full Text]
  29. Eiserich JP, Hristova M, Cross CE, Jones AD, Freeman BA, Halliwell B. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature. 1998;391:393–397.[Medline] [Order article via Infotrieve]
  30. Jiang Q, Hurst JK. Relative chlorinating, nitrating and oxidizing capabilities of neutrophil determined with phagocytosable probes. J Biol Chem. 1997;272:32767–32772.[Abstract/Free Full Text]
  31. Daugherty A, Dunn JL, Rateri DL, Heinecke JW. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions. J Clin Invest. 1994;94:437–444.
  32. Hazen SL, Crowley JR, Mueller DM, Heinecke JW. Mass spectrometric quantification of 3-chlorotyrosine in human tissues with attomole sensitivity: a sensitive and specific marker for myeloperoxidase-catalyzed chlorination at sites of inflammation. Free Radic Biol Med. 1997;23:909–916.[Medline] [Order article via Infotrieve]
  33. Fukuto JM. Chemistry of nitric oxide: biologically relevant aspects. In: Ignarro L, Murad F, eds. Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications. San Diego, Calif: Academic Press; 1995:1–16.
  34. Schmitt D, Shen Z, Zhang R, Colles SM, Wu W, Salomon RG, Chen Y, Chisolm GM, Hazen SL. Leukocytes utilize myeloperoxidase-generated nitrating intermediates as physiological catalysts for the generation of biologically active oxidized lipids and sterols in serum. Biochemistry. In press.
  35. Panasenko OM, Briviba K, Klotz LO, Sies H. Oxidative modification and nitration of human low-density lipoproteins by the reaction of hypochlorous acid with nitrite. Arch Biochem Biophys. 1997;343:254–259.[Medline] [Order article via Infotrieve]
  36. Markwell MA, Haas SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978;87:206–210.[Medline] [Order article via Infotrieve]
  37. Nelson DP, Kiesow LA. Enthalpy of decomposition of hydrogen peroxide by catalase at 25°C (with molar extinction coefficients of H2O2 solutions in the UV). Anal Biochem. 1972;49:474–478.[Medline] [Order article via Infotrieve]
  38. Schmidt HHHW, Kelm M. Determination of nitrite and nitrate by the Griess reaction. In: Feelisch M, Stamler JS, eds. Methods in Nitric Oxide Research. New York: John Wiley & Sons Ltd; 1996:491–497.
  39. Czerniecki BJ, Carter C, Rivoltini L, Koski GK, Kim HI, Weng DE, Roros JG, Hijazi YM, Xu S, Rosenberg SA, Cohen PA. Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells. J Immunol. 1997;159:3823–3837.[Abstract]
  40. Mooradian DL, Hutsell TC, Keefer LK. Nitric oxide (NO) donor molecules: effect of NO release rate on vascular smooth muscle cell proliferation in vitro. J Cardiovasc Pharmacol. 1995;25:674–678.[Medline] [Order article via Infotrieve]
  41. Baek KJ, Thiel BA, Lucas S, Stuehr DJ. Macrophage nitric oxide synthase subunits: purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. J Biol Chem. 1993;268:21120–21129.[Abstract/Free Full Text]
  42. Pratico D, Fitzgerald GA. Generation of 8-epiprostaglandin F2{alpha} by human monocytes: discriminate production by reactive oxygen species and prostaglandin endoperoxide synthase-2. J Biol Chem. 1996;271:8919–8924.[Abstract/Free Full Text]
  43. Hazen SL, Hsu FF, Gaut JP, Crowley JR, Heinecke JW. Modification of proteins and lipids by myeloperoxidase-derived oxidants. Methods Enzymol. 1999;300:88–105.[Medline] [Order article via Infotrieve]
  44. Crowley JR, Yarasheski K, Leeuwenburgh C, Turk J, Heinecke JW. Isotope dilution mass spectrometric quantification of 3-nitrotyrosine in proteins and tissues is facilitated by reduction to 3-aminotyrosine. Anal Biochem. 1998;259:127–135.[Medline] [Order article via Infotrieve]
  45. Hazen SL, Hsu FF, Mueller DM, Crowley JR, Heinecke JW. Human neutrophils employ chlorine gas as an oxidant during phagocytosis. J Clin Invest. 1996;98:1283–1289.[Medline] [Order article via Infotrieve]
  46. Savenkova ML, Mueller DM, Heinecke JW. Tyrosyl radical generated by myeloperoxidase is a physiological catalyst for the initiation of lipid peroxidation in low density lipoprotein. J Biol Chem. 1994;269:20394–20400.[Abstract/Free Full Text]
  47. Liu TZ, Stern A, Morrow JD. The isoprostanes: unique bioactive products of lipid peroxidation. An overview. J Biomed Sci. 1998;5:415–420.[Medline] [Order article via Infotrieve]
  48. Mallat Z, Nakamura T, Ohan J, Leseche G, Tedgui A, Maclouf J, Murphy RC. The relationship of hydroxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis. J Clin Invest. 1999;103:421–427.[Medline] [Order article via Infotrieve]
  49. Kettle AJ, Winterbourn CC. Myeloperoxidase: a key regulator of neutrophil oxidant production. Redox Rep. 1997;3:3–15.
  50. Fridovich I. CRC Handbook of Methods: Oxygen Radical Research. Boca Raton, Fla: CRC Press; 1985:213–215.
  51. Nelson SK, Bose SK, McCord JM. The toxicity of high-dose superoxide dismutase suggests that superoxide can both initiate and terminate lipid peroxidation in the reperfused heart. Free Radic Biol Med. 1994;16:195–200.[Medline] [Order article via Infotrieve]
  52. Test ST, Lampert MB, Ossanna PJ, Thoene JG, Weiss SJ. Generation of nitrogen-chlorine oxidants by human phagocytes. J Clin Invest. 1984;74:1341–1349.
  53. Lampert MB, Weiss SJ. The chlorinating potential of the human monocyte. Blood. 1983;62:645–651.