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• W2090819818 abstract "Nitric oxide, a pivotal molecule in vascular homeostasis, is converted under aerobic conditions to nitrite. Recent studies have shown that myeloperoxidase (MPO), an abundant heme protein released by activated leukocytes, can oxidize nitrite (NO2−) to a radical species, most likely nitrogen dioxide. Furthermore, hypochlorous acid (HOCl), the major strong oxidant generated by MPO in the presence of physiological concentrations of chloride ions, can also react with nitrite, forming the reactive intermediate nitryl chloride. Since MPO and MPO-derived HOCl, as well as reactive nitrogen species, have been implicated in the pathogenesis of atherosclerosis through oxidative modification of low density lipoprotein ( LDL ), we investigated the effects of physiological concentrations of nitrite (12.5–200 μm) on MPO-mediated modification of LDL in the absence and presence of physiological chloride concentrations. Interestingly, nitrite concentrations as low as 12.5 and 25 μmsignificantly decreased MPO/H2O2/Cl−-induced modification of apoB lysine residues, formation of N-chloramines, and increases in the relative electrophoretic mobility of LDL. In contrast, none of these markers of LDL atherogenic modification were affected by the MPO/H2O2/NO2−system. Furthermore, experiments using ascorbate (12.5–200 μm) and the tyrosine analogue 4-hydroxyphenylacetic acid (12.5–200 μm), which are both substrates of MPO, indicated that nitrite inhibits MPO-mediated LDL modifications by trapping the enzyme in its inactive compound II form. These data offer a novel mechanism for a potential antiatherogenic effect of the nitric oxide congener nitrite. Nitric oxide, a pivotal molecule in vascular homeostasis, is converted under aerobic conditions to nitrite. Recent studies have shown that myeloperoxidase (MPO), an abundant heme protein released by activated leukocytes, can oxidize nitrite (NO2−) to a radical species, most likely nitrogen dioxide. Furthermore, hypochlorous acid (HOCl), the major strong oxidant generated by MPO in the presence of physiological concentrations of chloride ions, can also react with nitrite, forming the reactive intermediate nitryl chloride. Since MPO and MPO-derived HOCl, as well as reactive nitrogen species, have been implicated in the pathogenesis of atherosclerosis through oxidative modification of low density lipoprotein ( LDL ), we investigated the effects of physiological concentrations of nitrite (12.5–200 μm) on MPO-mediated modification of LDL in the absence and presence of physiological chloride concentrations. Interestingly, nitrite concentrations as low as 12.5 and 25 μmsignificantly decreased MPO/H2O2/Cl−-induced modification of apoB lysine residues, formation of N-chloramines, and increases in the relative electrophoretic mobility of LDL. In contrast, none of these markers of LDL atherogenic modification were affected by the MPO/H2O2/NO2−system. Furthermore, experiments using ascorbate (12.5–200 μm) and the tyrosine analogue 4-hydroxyphenylacetic acid (12.5–200 μm), which are both substrates of MPO, indicated that nitrite inhibits MPO-mediated LDL modifications by trapping the enzyme in its inactive compound II form. These data offer a novel mechanism for a potential antiatherogenic effect of the nitric oxide congener nitrite. nitric-oxide synthase apolipoprotein B-100 diethylenetriaminepentaacetic acid 4-hydroxyphenylacetic acid low density lipoprotein myeloperoxidase phosphate-buffered saline (10 mm sodium phosphate, 140 mm NaCl, pH 7.4) relative electrophoretic mobility thionitrobenzoic acid: HPLC, high performance liquid chromatography Nitric oxide (nitrogen monoxide, NO⋅) is synthesizedin vivo by a family of inducible and constitutively expressed nitric-oxide synthases (NOS)1 (1Moncada S. Higgs A. N. Engl. J. Med. 1993; 329: 2002-2012Crossref PubMed Scopus (5661) Google Scholar, 2Ignarro L.J. FASEB J. 1989; 3: 31-36Crossref PubMed Scopus (569) Google Scholar). Nitric oxide generated by the NOS isoform present in endothelial cells (eNOS) is critically involved in normal vascular function through regulation of smooth muscle cell relaxation and vasodilation as well as modulation of platelet, leukocyte, and endothelial cell adhesion (1Moncada S. Higgs A. N. Engl. J. Med. 1993; 329: 2002-2012Crossref PubMed Scopus (5661) Google Scholar, 2Ignarro L.J. FASEB J. 1989; 3: 31-36Crossref PubMed Scopus (569) Google Scholar). The inducible NOS isoform present in phagocytes (iNOS) is thought to be involved in their antimicrobial activity, whereas up-regulation of iNOS during chronic inflammation has been implicated in vascular pathology (1Moncada S. Higgs A. N. Engl. J. Med. 1993; 329: 2002-2012Crossref PubMed Scopus (5661) Google Scholar, 3Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar). Since nitric oxide does not readily react with biological macromolecules, the tissue damage associated with increased nitric oxide levels has been attributed to the generation of peroxynitrite (3Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar), which is formed by rapid reaction of nitric oxide with superoxide (k 2 = 1.9 × 1010m−1s−1) (4Koppenol W.H. Free Radic. Biol. Med. 1998; 25: 385-391Crossref PubMed Scopus (303) Google Scholar). Under aerobic conditions, nitric oxide also reacts with molecular oxygen (k 2 = 6 × 106m−2s−1) (5Wink D.A. Darbyshire R.W. Nims J.E. Sasvedra J.E. Ford P.C. Chem. Res. Toxicol. 1993; 6: 23-27Crossref PubMed Scopus (478) Google Scholar) to form a dinitrogen trioxide intermediate that hydrolyzes to nitrite (Reaction 1) (5Wink D.A. Darbyshire R.W. Nims J.E. Sasvedra J.E. Ford P.C. Chem. Res. Toxicol. 1993; 6: 23-27Crossref PubMed Scopus (478) Google Scholar, 6Ignarro L.J. Fukuto J.M. Griscavage J.M. Rogers N.E. Byrns R.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8103-8107Crossref PubMed Scopus (742) Google Scholar, 7Kharitonov V.G. Sundquist A.R. Sharma V.S. J. Biol. Chem. 1994; 269: 5881-5883Abstract Full Text PDF PubMed Google Scholar). Nitrite is found in biological fluids at concentrations between 0.5 and 210 μm (8Leone A.M. Francis P.L. Rhodes P. Moncada S. Biochem. Biophys. Res. Commun. 1994; 200: 951-957Crossref PubMed Scopus (192) Google Scholar, 9Ueda T. Maekawa T. Sadamitsu D. Oshita S. Ogino K. Nakamura K. Electrophoresis. 1995; 16: 1002-1004Crossref PubMed Scopus (85) Google Scholar, 10Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10586) Google Scholar). In human plasma, levels of nitrite are typically low, between 0.5 and 3.3 μm (8Leone A.M. Francis P.L. Rhodes P. Moncada S. Biochem. Biophys. Res. Commun. 1994; 200: 951-957Crossref PubMed Scopus (192) Google Scholar, 9Ueda T. Maekawa T. Sadamitsu D. Oshita S. Ogino K. Nakamura K. Electrophoresis. 1995; 16: 1002-1004Crossref PubMed Scopus (85) Google Scholar), due to oxidation of nitrite to nitrate by oxyhemoglobin (3Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar). In inflammatory conditions, however, plasma nitrite levels can significantly increase,e.g. up to 36 μm in patients with human immunodeficiency virus infection (11Torre D. Ferrario G. Speranza F. Orani A. Fiori G.P. Zeroli C. J. Clin. Pathol. 1996; 49: 574-576Crossref PubMed Scopus (70) Google Scholar).2NO⋅+½O2→N2O3→H2O2NO2−+2H+REACTION1Leukocytes such as neutrophils, monocytes, and macrophages, as well as endothelial cells, can synthesize both nitric oxide and superoxide (1Moncada S. Higgs A. N. Engl. J. Med. 1993; 329: 2002-2012Crossref PubMed Scopus (5661) Google Scholar, 12Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3812) Google Scholar). Thus, it is likely that peroxynitrite is formed in vivo by these cells. 3-Nitrotyrosine, a product of the reaction of peroxynitrite with either free tyrosine or tyrosine residues in (lipo)proteins has been used as a biomarker for the generation of peroxynitrite in vivo (13Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (904) Google Scholar). Elevated 3-nitrotyrosine levels have been detected, e.g. in atherosclerotic lesions (14Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1067) Google Scholar, 15Leeuwenburgh C. Hardy M.M. Hazen S.L. Wagner P. Oh-ishi S. Steinbrecher U.P. Heinecke J.W. J. Biol. Chem. 1997; 272: 1433-1436Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar), as has increased expression of the inducible NOS isoform (16Buttery L.D. Springall D.R. Chester A.H. Evans T.J. Standfield E.N. Parums D.V. Yacoub M.H. Polak J.M. Lab. Invest. 1996; 75: 77-85PubMed Google Scholar, 17Luoma J.S. Stralin P. Marklund S.L. Hiltunen T.P. Sarkioja T. Yla-Herttuala S. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 157-167Crossref PubMed Scopus (227) Google Scholar). Leukocytes, however, also release the abundant heme protein myeloperoxidase (MPO) upon activation by inflammatory stimuli (12Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3812) Google Scholar, 18Kettle A.J. Winterbourn C.C. Redox Rep. 1997; 3: 3-15Crossref PubMed Scopus (578) Google Scholar), and recent studies show that mammalian peroxidases can oxidize nitrite to a radical species, most likely nitrogen dioxide (Reaction 2) (19Reszka K.J. Matuszak Z. Chignell C.F. Dillon J. Free. Radic. Biol. Med. 1999; 26: 669-678Crossref PubMed Scopus (44) Google Scholar, 20Burner U. Furtmuller P.G. Kettle A.J. Koppenol W.H. Obinger C. J. Biol. Chem. 2000; 275: 20597-20601Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Furthermore, hypochlorous acid (HOCl), the major strong oxidant generated by MPO in the presence of physiological concentrations of chloride ions (Reaction 3) (12Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3812) Google Scholar, 18Kettle A.J. Winterbourn C.C. Redox Rep. 1997; 3: 3-15Crossref PubMed Scopus (578) Google Scholar), can also react with nitrite, forming the reactive intermediate nitryl chloride (Reaction 4) (21Eiserich J.P. Cross C.E. Jones A.D. Halliwell B.,. van der Vliet A. J. Biol. Chem. 1996; 271: 19199-19208Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 22Panasenko O.M. Briviba K. Klotz L. Sies H. Arch. Biochem. Biophys. 1997; 343: 254-259Crossref PubMed Scopus (88) Google Scholar). Both nitrogen dioxide and nitryl chloride, like peroxynitrite, can nitrate tyrosine residues (21Eiserich J.P. Cross C.E. Jones A.D. Halliwell B.,. van der Vliet A. J. Biol. Chem. 1996; 271: 19199-19208Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 23van der Vliet A. Eiserich J.P. Halliwell B. Cross C.E. J. Biol. Chem. 1997; 272: 7617-7625Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar,24Sampson J.B. Ye Y. Rosen H. Beckman J.S. Arch. Biochem. Biophys. 1998; 356: 207-213Crossref PubMed Scopus (295) Google Scholar), calling into question the specificity of 3-nitrotyrosine as a marker of peroxynitrite generation in vivo (25Halliwell B. FEBS Lett. 1997; 411: 157-160Crossref PubMed Scopus (433) Google Scholar, 26Kettle A.J. Van Dalen C.J. Winterbourn C.C. Redox Rep. 1997; 3: 257-258Crossref PubMed Scopus (84) Google Scholar).2NO2−+H2O2+2H+→MPO2NO2⋅+2H2OREACTION2Cl−+H2O2+H+→MPOHOCl+H2OREACTION3NO2−+HOCl+H+→Cl­NO2+H2OREACTION4Several recent studies implicate MPO and MPO-derived HOCl in the pathogenesis of atherosclerosis (27Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1103) Google Scholar, 28Hazell L.J. Arnold L. Flowers D. Waeg G. Malle E. Stocker R. J. Clin. Invest. 1996; 97: 1535-1544Crossref PubMed Scopus (529) Google Scholar, 29Malle E. Waeg G. Schreiber R. Grone E.F. Sattler W. Grone H.J. Eur. J. Biochem. 2000; 267: 4495-4503Crossref PubMed Scopus (216) Google Scholar, 30Leeuwenburgh C. Rasmussen J.E. Hsu F.F. Mueller D.M. Pennathur S. Heinecke J.W. J. Biol. Chem. 1997; 272: 3520-3526Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 31Hazen S.L. Heinecke J.W. J. Clin. Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (744) Google Scholar). Catalytically active MPO (27Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1103) Google Scholar) and epitopes recognized by antibodies against HOCl-modified proteins have been detected in human atherosclerotic lesions (28Hazell L.J. Arnold L. Flowers D. Waeg G. Malle E. Stocker R. J. Clin. Invest. 1996; 97: 1535-1544Crossref PubMed Scopus (529) Google Scholar); these were found to colocalize with monocyte/macrophages, endothelial cells, and the extracellular matrix (29Malle E. Waeg G. Schreiber R. Grone E.F. Sattler W. Grone H.J. Eur. J. Biochem. 2000; 267: 4495-4503Crossref PubMed Scopus (216) Google Scholar). Dityrosine and 3-chlorotyrosine, biomarkers of MPO- and HOCl-mediated protein modification, have also been detected in atherosclerotic lesions (30Leeuwenburgh C. Rasmussen J.E. Hsu F.F. Mueller D.M. Pennathur S. Heinecke J.W. J. Biol. Chem. 1997; 272: 3520-3526Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 31Hazen S.L. Heinecke J.W. J. Clin. Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (744) Google Scholar). Oxidative modification of low density lipoprotein (LDL) in vitro by MPO or HOCl primarily involves chlorination of the ε-amino groups of lysine residues of apolipoprotein B-100 (apoB), the major protein component of LDL, resulting in the formation ofN-chloramines (32Hazell L.J. Stocker R. Biochem. J. 1993; 290: 165-172Crossref PubMed Scopus (293) Google Scholar, 33Hazell L.J. van den Berg J.J. Stocker R. Biochem. J. 1994; 302: 297-304Crossref PubMed Scopus (243) Google Scholar, 34Carr A.C. Tijerina T. Frei B. Biochem. J. 2000; 346: 491-499Crossref PubMed Scopus (93) Google Scholar). LDL-associatedN-chloramines have been implicated in the altered electrophoretic migration, aggregation, and subsequent uncontrolled uptake of HOCl-modified LDL by macrophages (32Hazell L.J. Stocker R. Biochem. J. 1993; 290: 165-172Crossref PubMed Scopus (293) Google Scholar, 33Hazell L.J. van den Berg J.J. Stocker R. Biochem. J. 1994; 302: 297-304Crossref PubMed Scopus (243) Google Scholar, 35Ryu B.H. Mao F.W. Lou P. Gutman R.L. Greenspan P. Biosci. Biotechnol. Biochem. 1995; 59: 1619-1622Crossref PubMed Scopus (27) Google Scholar). Since 3-nitrotyrosine has also been detected in atherosclerotic lesions (14Beckman J.S. Ye Y.Z. Anderson P.G. Chen J. Accavitti M.A. Tarpey M.M. White C.R. Biol. Chem. Hoppe-Seyler. 1994; 375: 81-88Crossref PubMed Scopus (1067) Google Scholar,15Leeuwenburgh C. Hardy M.M. Hazen S.L. Wagner P. Oh-ishi S. Steinbrecher U.P. Heinecke J.W. J. Biol. Chem. 1997; 272: 1433-1436Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar), it is possible that MPO-derived reactive nitrogen species are involved in atherogenesis in addition to HOCl. In contrast to HOCl, however, MPO-derived reactive nitrogen species primarily cause lipid peroxidation in LDL (36Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (416) Google Scholar, 37Byun J. Mueller D.M. Fabjan J.S. Heinecke J.W. FEBS Lett. 1999; 455: 243-246Crossref PubMed Scopus (106) Google Scholar, 38Hazen S.L. Zhang R. Shen Z. Wu W. Podrez E.A. MacPherson J.C. Schmitt D. Mitra S.N. Mukhopadhyay C. Chen Y. Cohen P.A. Hoff H.F. Abu-Soud H.M. Circ. Res. 1999; 85: 950-958Crossref PubMed Scopus (200) Google Scholar). Thus, increased macrophage uptake of LDL modified by reactive nitrogen species is most likely due to increased levels of lipid oxidation products (36Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (416) Google Scholar, 39Podrez E.A. Febbraio M. Sheibani N. Schmitt D. Silverstein R.L. Hajjar D.P. Cohen P.A. Frazier W.A. Hoff H.F. Hazen S.L. J. Clin. Invest. 2000; 105: 1095-1108Crossref PubMed Scopus (359) Google Scholar). In this study we investigated the effects of physiological concentrations of nitrite (12.5–200 μm) on MPO-mediated modification of LDL in the absence and presence of physiological chloride concentrations (140 mm). In addition, we examined whether the physiological antioxidant ascorbate can inhibit LDL modification under these conditions. Our data reveal a novel mechanism by which nitrite inhibits MPO-mediated atherogenic modification of LDL. Human leukocyte MPO and anti-nitrotyrosine monoclonal antibodies were procured from Calbiochem, HOCl was from Aldrich, and 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) was from Molecular Probes, Eugene, OR. Alkaline phosphatase-conjugated anti-mouse IgG antibody and BCIP/TNBT color reagent were from Chemicon International, Temecula, CA. All other reagents were obtained from Sigma. Phosphate-buffered saline (PBS) was composed of 10 mm sodium phosphate buffer, 140 mm NaCl, pH 7.4, and contained the metal chelator diethylenetriaminepentaacetic acid (DTPA, 100 μm). Tris-buffered saline was composed of 10 mm Tris-HCl, 140 mm NaCl, pH 7.4, and contained 0.1% Tween 20. Griess reagent was composed of 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4. Thionitrobenzoic acid (TNB) was prepared from 5,5′-dithiobis-(2-nitrobenzoic acid) by alkaline hydrolysis with subsequent neutralization (40Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (224) Google Scholar). LDL was isolated from fresh plasma by a sequential centrifugation method (41Sattler W. Mohr D. Stocker R. Methods Enzymol. 1994; 233: 469-489Crossref PubMed Scopus (283) Google Scholar) modified as described by us previously (34Carr A.C. Tijerina T. Frei B. Biochem. J. 2000; 346: 491-499Crossref PubMed Scopus (93) Google Scholar). The isolated LDL (1.019 < d < 1.067 g/ml fraction) was desalted by two sequential passages through PD-10 gel filtration columns (Amersham Pharmacia Biotech) using 10 mm sodium phosphate buffer, pH 7.4. The total protein was estimated using the Lowry micro method kit (Sigma P5656). For experiments the LDL was diluted to a concentration of 0.5 mg of protein/ml (≈1 μm LDL) in PBS unless indicated otherwise. For chloride-free experiments, the LDL was diluted in phosphate buffer containing 100 μm DTPA. MPO (50 nm) was incubated with LDL for 30 min at 37 °C in the presence of the stated concentrations of H2O2. The H2O2 was freshly diluted in phosphate buffer and standardized at 240 nm (ε = 43.6m−1 cm−1) (42Kettle A.J. Winterbourn C.C. Biochem. J. 1988; 252: 529-536Crossref PubMed Scopus (149) Google Scholar) and was added to the LDL samples in aliquots of 25–50 μm over the 30-min incubation period. Under these conditions all of the added H2O2 was converted into HOCl as determined by TNB oxidation using 10 mmtaurine as a trap (40Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (224) Google Scholar). LDL samples were also incubated in the presence of the MPO substrates nitrite, ascorbate, and 4-hydroxyphenylacetic acid (HPA) (12.5 - 200 μm each). HPA was used instead of tyrosine because it lacks an amino group and, thus, has only minimal reactivity with HOCl. Ascorbate was freshly diluted in phosphate buffer containing 100 μm DTPA and standardized at 265 nm (ε = 15,000m−1 cm−1) (43Chesney J.A. Mahoney J.R. Eaton J.W. Anal. Biochem. 1991; 196: 262-266Crossref PubMed Scopus (36) Google Scholar). Ascorbate was measured using paired-ion, reversed-phase HPLC with electrochemical detection (44Frei B. England L. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6377-6381Crossref PubMed Scopus (1688) Google Scholar). Nitrite was measured spectrophotometrically at 550 nm using the Griess reagent (45Vodovotz Y. Biotechniques. 1996; 20: 390-394Crossref PubMed Scopus (69) Google Scholar). HOCl was standardized at 292 nm (ε = 350 m−1cm−1) after dilution into pH 12 buffer (46Morris J.C. J. Phys. Chem. 1966; 70: 3798-3805Crossref Scopus (796) Google Scholar). Standardized HOCl was freshly diluted in PBS; the pK a of HOCl is 7.5 (46Morris J.C. J. Phys. Chem. 1966; 70: 3798-3805Crossref Scopus (796) Google Scholar), and therefore the solution contained both HOCl and OCl−. HOCl was added to the LDL in the presence of nitrite (coincubation) or before the addition of nitrite (pre-incubation), and the LDL was incubated for 30 min at 37 °C. Tryptophan residues were measured directly by fluorescence (excitation = 280 nm, emission = 335 nm) (32Hazell L.J. Stocker R. Biochem. J. 1993; 290: 165-172Crossref PubMed Scopus (293) Google Scholar). In samples containing HPA, the fluorescence of the equivalent concentration of HPA was subtracted from the total fluorescence. Lysine residues were measured after fluorescamine derivatization (excitation = 390 nm, emission = 475 nm) (47Bohlen P. Stein S. Dairman W. Udenfriend S. Arch. Biochem. Biophys. 1973; 155: 213-220Crossref PubMed Scopus (1329) Google Scholar). Cysteine residues were measured after 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) derivatization (excitation = 365 nm, emission = 490 nm) (48Toyo'oka T. Imai K. Anal. Chem. 1984; 56: 2461-2464Crossref Scopus (156) Google Scholar), as described by us previously (34Carr A.C. Tijerina T. Frei B. Biochem. J. 2000; 346: 491-499Crossref PubMed Scopus (93) Google Scholar). N-Chloramines were determined after TNB oxidation (ε412 = 14, 100m−1 cm−1) (40Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (224) Google Scholar). Carbonyls were measured after 2,4-dinitrophenylhydrazine derivatization (ε450 = 22,000m−1 cm−1) (33Hazell L.J. van den Berg J.J. Stocker R. Biochem. J. 1994; 302: 297-304Crossref PubMed Scopus (243) Google Scholar). 3-Nitrotyrosine was measured by dot blot using an anti-nitrotyrosine monoclonal antibody (49Ye Y.Z. Strong M. Huang Z.Q. Beckman J.S. Methods Enzymol. 1996; 269: 201-209Crossref PubMed Google Scholar). Briefly, nitrocellulose blots were treated for 1 h with 5% milk powder in Tris-buffered saline, 1 h with anti-nitrotyrosine monoclonal antibody (1:200 in Tris-buffered saline), 1 h with alkaline phosphatase-conjugated anti-mouse IgG antibody (1:5,000 in Tris-buffered saline) and detected with BCIP/TNBT color reagent. The change in the relative electrophoretic mobility (REM) of LDL was determined by agarose gel electrophoresis using a Paragon lipoprotein electrophoresis kit (Beckman Coulter, Fullerton, CA). Statistical analyses were carried out using analysis of variance with Fisher's post hoc analysis and regression analysis using StatView software (SAS Institute, Cary, NC). Statistical significance was set at p < 0.05. We (34Carr A.C. Tijerina T. Frei B. Biochem. J. 2000; 346: 491-499Crossref PubMed Scopus (93) Google Scholar) and others (32Hazell L.J. Stocker R. Biochem. J. 1993; 290: 165-172Crossref PubMed Scopus (293) Google Scholar, 33Hazell L.J. van den Berg J.J. Stocker R. Biochem. J. 1994; 302: 297-304Crossref PubMed Scopus (243) Google Scholar) have shown that bolus addition of reagent HOCl (25–200 μm) to human LDL results in a number of modifications to apoB, e.g. oxidation of cysteine, tryptophan, and lysine residues, formation of N-chloramines, and an increase in REM. In the present study, we investigated the physiologically more relevant system of HOCl generated by MPO/H2O2/Cl−. The addition of increasing amounts of H2O2 (25–200 μm) to human LDL (0.5 mg protein/ml; ≈1 μm) in the presence of MPO (50 nm) caused basically identical modifications to LDL (Fig.1) as those seen with reagent HOCl (34Carr A.C. Tijerina T. Frei B. Biochem. J. 2000; 346: 491-499Crossref PubMed Scopus (93) Google Scholar). In contrast, 200 μm H2O2 alone did not cause oxidation of LDL amino acid residues or an increase in REM (data not shown). Using the MPO/H2O2/Cl− system, all of the added H2O2 was converted into HOCl as determined by TNB oxidation using taurine as a trap (40Kettle A.J. Winterbourn C.C. Methods Enzymol. 1994; 233: 502-512Crossref PubMed Scopus (224) Google Scholar). Cysteine residues were the most sensitive target on LDL and were completely oxidized in the presence of 75 μmH2O2 (Fig. 1 a). Lysine and tryptophan residues were modified at equal rates, with 39 and 43% of the residues modified, respectively, after the addition of 200 μm H2O2 (Fig. 1 a). Since LDL contains ∼3–5 free cysteine residues (50Sommer A. Gorges R. Kostner G.M. Paltauf F. Hermetter A. Biochemistry. 1991; 30: 11245-11249Crossref PubMed Scopus (54) Google Scholar, 51Yang C.Y. Kim T.W. Weng S.A. Lee B.R. Yang M.L. Gotto A.M.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5523-5527Crossref PubMed Scopus (82) Google Scholar), 356 lysine residues, and 37 tryptophan residues (52Esterbauer H. Gebicki J. Puhl H. Jurgens G. Free Radic. Biol. Med. 1992; 13: 341-390Crossref PubMed Scopus (2128) Google Scholar), lysine residues can be calculated to be quantitatively the major target of HOCl generated by the MPO system. The decrease in lysine residues was mirrored by the formation of N-chloramines, which accounted for approximately one-third of the added H2O2 (Fig.1 b). A dose-dependent increase in REM was also observed with increasing concentrations of H2O2(Fig. 1 b). Previous studies have investigated the modification of LDL by MPO/H2O2/NO2−in the absence of chloride (36Podrez E.A. Schmitt D. Hoff H.F. Hazen S.L. J. Clin. Invest. 1999; 103: 1547-1560Crossref PubMed Scopus (416) Google Scholar, 37Byun J. Mueller D.M. Fabjan J.S. Heinecke J.W. FEBS Lett. 1999; 455: 243-246Crossref PubMed Scopus (106) Google Scholar). We found that this system caused relatively few changes to apoB under the conditions used in the present study (Fig. 2). No significant modification of lysine residues occurred, and only a small dose-dependent decrease of tryptophan residues was observed with increasing nitrite concentrations (Fig. 2 a); ∼25% of the tryptophan residues in LDL were oxidized in the presence of 200 μm nitrite. Analysis of cysteine residues was confounded by the fact that in the absence of nitrite ∼50% of the residues were already oxidized (Fig. 2 a). Since 200 μmH2O2 alone did not cause cysteine oxidation (data not shown), this finding is most likely due to a small amount of halide contamination in the buffers used. Nevertheless, nitrite did not exert any dose-dependent effect on oxidation of apoB cysteine residues (Fig. 2 a). In agreement with the lack of lysine oxidation by the MPO/H2O2/NO2−system, there was no increase in the REM or TNB reactivity of LDL (Fig.2 c). However, dot blot analysis using an anti-nitrotyrosine monoclonal antibody showed dose-dependent formation of 3-nitrotyrosine (Fig. 2 b). Interestingly, measurement of nitrite using the Griess assay indicated that approximately twice as much of the added nitrite was consumed in the absence of chloride than in its presence (e.g. 124 ± 4 μm nitriteversus 56 ± 7 μm nitrite, respectively, after the addition of 200 μm nitrite) (data not shown). It should be noted, however, that reaction of nitrogen dioxide with substrates that can donate hydrogens regenerates nitrite, and thus, the Griess assay may underestimate the amount of nitrite utilized by MPO. Since nitrite is a substrate for MPO (20Burner U. Furtmuller P.G. Kettle A.J. Koppenol W.H. Obinger C. J. Biol. Chem. 2000; 275: 20597-20601Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), the effect of physiological concentrations of nitrite on LDL modification in the presence of 200 μmH2O2 and physiological chloride concentrations was investigated. The addition of increasing concentrations of nitrite (12.5–200 μm) affected specific MPO-mediated modifications of LDL (Fig. 3). Low concentrations of nitrite (≥12.5 μm) significantly decreased modification of apoB lysine residues induced by MPO/H2O2/Cl− (Fig. 3 a). A concurrent, although less extensive, decrease in modification of tryptophan residues was observed at 25–50 μm nitrite, but this effect was lost at higher nitrite concentrations (Fig.3 a). Since the cysteine residues were almost fully oxidized with 200 μm H2O2 (Fig.3 a), LDL was treated with sufficient H2O2 (25 μm) to oxidize approximately one-third of the cysteine residues; the addition of nitrite, however, did not have a significant effect on thiol oxidation at this concentration of H2O2 (data not shown). In agreement with the decrease in lysine oxidation by low concentrations of nitrite (Fig. 3 a), formation ofN-chloramines and increase in REM were also inhibited (Fig.3 b). Interestingly, at higher concentrations of nitrite (150 and 200 μm), the REM increased again, although not to control levels seen in the absence of nitrite (Fig. 3 b). This increase in REM could be a result of increased nitrite-dependent lipid peroxidation and subsequent derivatization of apoB lysine residues to carbonyls by lipid hydroperoxide breakdown products. In support of this hypothesis, we found that nitrite dose-dependently increased apoB carbonyl levels (e.g. 14 ± 1 μm carbonyls without nitrite versus 60 ± 2 μm carbonyls at 200 μm nitrite) but not until the concentrations of added nitrite reached ≥50 μm (data not shown). A number of mechanisms could account for the marked decrease in lysine oxidation,N-chloramine formation, and increase in REM by MPO/H2O2/Cl− in the presence of low concentrations of nitrite (Figs. 3, a and b). For example, nitrite could “scavenge” MPO-derived HOCl (21Eiserich J.P. Cross C.E. Jones A.D. Halliwell B.,. van der Vliet A. J. Biol. Chem. 1996; 271: 19199-19208Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 22Panasenko O.M. Briviba K. Klotz L. Sies H. Arch. Biochem. Biophys. 1997; 343: 254-259Crossref PubMed Scopus (88) Google Scholar) or react with preformed N-chloramines. To investigate the former mechani" @default.
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• W2090819818 title "The Nitric Oxide Congener Nitrite Inhibits Myeloperoxidase/H2O2/ Cl−-mediated Modification of Low Density Lipoprotein" @default.
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