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• W1976760968 abstract "Recently, it was proposed that neutrophils generate ozone (Wentworth, P. J., McDunn, J. E., Wentworth, A. D., Takeuchi, C., Nieva, J., Jones, T., Bautista, C., Ruedi, J. M., Gutierrez, A., Janda, K. D., Babior, B. M., Eschenmoser, A., and Lerner, R. A. (2002) Science 298, 2195–2199; Babior, B. M., Takeuchi, C., Ruedi, J., Gutierrez, A., and Wentworth, P. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 3031–3034). Evidence for the proposal was based largely on the chemistry of ozone reacting with indigo carmine to produce isatin sulfonic acid. In this investigation, we have examined the specificity of this reaction and whether it can be used as unequivocal evidence of ozone production by neutrophils. Stimulated neutrophils promoted the loss of indigo carmine and formation of isatin sulfonic acid in a reaction that was completely inhibited by superoxide dismutase. Methionine, which scavenges ozone, singlet oxygen, and hypochlorous acid, had no effect on the reaction. Neither did catalase or azide, which scavenge hydrogen peroxide and inhibit myeloperoxidase, respectively. From these results, it is apparent that superoxide was responsible for bleaching indigo carmine. Superoxide generated using xanthine oxidase and acetaldehyde also converted indigo carmine to isatin sulfonic acid in a reaction that was completely inhibited by superoxide dismutase and unaffected by catalase. When the xanthine oxidase reaction was carried out in H218O, the proportion of 18O incorporated into the isatin sulfonic acid was the same as that found for ozone. Thus, reactions of ozone and superoxide with indigo carmine are indistinguishable with respect to isatin sulfonic acid formation. We conclude that bleaching of indigo carmine cannot be used to invoke ozone production by neutrophils. Studies using indigo carmine to implicate ozone in other biological processes should also be interpreted with caution. Recently, it was proposed that neutrophils generate ozone (Wentworth, P. J., McDunn, J. E., Wentworth, A. D., Takeuchi, C., Nieva, J., Jones, T., Bautista, C., Ruedi, J. M., Gutierrez, A., Janda, K. D., Babior, B. M., Eschenmoser, A., and Lerner, R. A. (2002) Science 298, 2195–2199; Babior, B. M., Takeuchi, C., Ruedi, J., Gutierrez, A., and Wentworth, P. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 3031–3034). Evidence for the proposal was based largely on the chemistry of ozone reacting with indigo carmine to produce isatin sulfonic acid. In this investigation, we have examined the specificity of this reaction and whether it can be used as unequivocal evidence of ozone production by neutrophils. Stimulated neutrophils promoted the loss of indigo carmine and formation of isatin sulfonic acid in a reaction that was completely inhibited by superoxide dismutase. Methionine, which scavenges ozone, singlet oxygen, and hypochlorous acid, had no effect on the reaction. Neither did catalase or azide, which scavenge hydrogen peroxide and inhibit myeloperoxidase, respectively. From these results, it is apparent that superoxide was responsible for bleaching indigo carmine. Superoxide generated using xanthine oxidase and acetaldehyde also converted indigo carmine to isatin sulfonic acid in a reaction that was completely inhibited by superoxide dismutase and unaffected by catalase. When the xanthine oxidase reaction was carried out in H218O, the proportion of 18O incorporated into the isatin sulfonic acid was the same as that found for ozone. Thus, reactions of ozone and superoxide with indigo carmine are indistinguishable with respect to isatin sulfonic acid formation. We conclude that bleaching of indigo carmine cannot be used to invoke ozone production by neutrophils. Studies using indigo carmine to implicate ozone in other biological processes should also be interpreted with caution. Neutrophils are essential for effective host defense against a wide range of bacteria and fungi (1Klebanoff S.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. Lippincott Williams & Wilkins, Philadelphia1999: 721-768Google Scholar, 2Hampton M.B. Kettle A.J. Winterbourn C.C. Blood. 1998; 92: 3007-3017Google Scholar). Although their oxidants have been intensely studied since Babior et al. (3Babior B.M. Curnutte J.T. Kipnes R.S. J. Lab. Clin. Med. 1975; 85: 235-244Google Scholar) found that neutrophils generate superoxide, a clear picture of how oxidants kill microorganisms has yet to emerge. Recently, it was proposed that neutrophils generate ozone and that this toxic oxidant contributes to the antimicrobial and inflammatory actions of these cells (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar, 5Wentworth P.J. Nieva J. Takeuchi C. Galve R. Wentworth A.D. Dilley R.B. DeLaria G.A. Saven A. Babior B.M. Janda K.D. Eschenmoser A. Lerner R.A. Science. 2003; 302: 1053-1056Google Scholar, 6Babior B.M. Takeuchi C. Ruedi J. Gutierrez A. Wentworth P.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3031-3034Google Scholar). This proposal challenges the contemporary paradigms used to explain oxidative killing of phagocytosed microorganisms. Several commentaries have been written on these findings, some proposing that the ways phagocytes employ their oxidants needs to be reevaluated (7Nathan C. Science. 2002; 298: 2143-2144Google Scholar, 8Lerner R.A. Eschenmoser A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3013-3015Google Scholar) and others questioning whether this is justified on the strength of the current evidence (9Parren P.W. Leusen J.H. van de Winkel J.G. Trends Immunol. 2003; 24: 467-469Google Scholar). Before ozone can be accepted as an important oxidant in the antimicrobial armory of the neutrophil, the mechanism by which it is generated and the evidence to support it needs further evaluation. Wentworth et al. (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar, 10Wentworth P.J. Jones L.H. Wentworth A.D. Zhu X. Larsen N.A. Wilson I.A. Xu X. Goddard W.A. Janda K.D. Eschenmoser A. Lerner R.A. Science. 2001; 293: 1806-1811Google Scholar) proposed a mechanism for ozone production, based on previous observations by this group on the antibody-catalyzed oxidation of water by singlet oxygen. It requires antibodies to be bound to the plasma membrane of the neutrophil and singlet oxygen to be generated by the cells. However, this remains speculative as it was not possible to suppress the reaction by antibody removal (6Babior B.M. Takeuchi C. Ruedi J. Gutierrez A. Wentworth P.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3031-3034Google Scholar), and the amounts that could be detected were much less than those needed to kill bacteria in a cell-free singlet oxygen/antibody system (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar). Whether neutrophils produce significant amounts of singlet oxygen is also debatable. Singlet oxygen can be produced by the reaction of hypochlorous acid with hydrogen peroxide (11Kanofsky J.R. Chem. Biol. Interact. 1989; 70: 1-28Google Scholar), both major products of the respiratory burst of neutrophils. When these cells are stimulated, they reduce molecular oxygen to superoxide, which dismutates to hydrogen peroxide (12Babior B.M. Blood. 1999; 93: 1464-1476Google Scholar). The neutrophil enzyme myeloperoxidase uses hydrogen peroxide to oxidize chloride to hypochlorous acid (13Kettle A.J. Winterbourn C.C. Redox Report. 1997; 3: 3-15Google Scholar). However, the reaction between hypochlorous acid and hydrogen peroxide requires an acid pH (14Kanofsky J.R. Wright J. Miles-Richardson G.E. Tauber A.I. J. Clin. Invest. 1984; 74: 1489-1495Google Scholar), in which neutrophil oxidase activity is suppressed (15Gabig T.G. Bearman S.I. Babior B.M. Blood. 1979; 53: 1133-1139Google Scholar); under physiological conditions, other reactions of hydrogen peroxide and hypochlorous acid are much more favored (2Hampton M.B. Kettle A.J. Winterbourn C.C. Blood. 1998; 92: 3007-3017Google Scholar). With some exceptions (16Tatsuzawa H. Maruyama T. Hori K. Sano Y. Nakano M. Biochem. Biophys. Res. Commun. 1999; 262: 647-650Google Scholar, 17Steinbeck M.J. Khan A.U. Karnovsky M.J. J. Biol. Chem. 1992; 267: 13425-13433Google Scholar), most studies have found low or undetectable singlet oxygen production from myeloperoxidase systems or neutrophils (1Klebanoff S.J. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. Lippincott Williams & Wilkins, Philadelphia1999: 721-768Google Scholar, 11Kanofsky J.R. Chem. Biol. Interact. 1989; 70: 1-28Google Scholar, 13Kettle A.J. Winterbourn C.C. Redox Report. 1997; 3: 3-15Google Scholar, 14Kanofsky J.R. Wright J. Miles-Richardson G.E. Tauber A.I. J. Clin. Invest. 1984; 74: 1489-1495Google Scholar), and the majority opinion is that it is not formed by these cells in appreciable amounts. The major evidence presented for ozone formation by neutrophils was that these cells converted indigo carmine to isatin sulfonic acid (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar, 6Babior B.M. Takeuchi C. Ruedi J. Gutierrez A. Wentworth P.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3031-3034Google Scholar). Ozone reacts rapidly with indigo carmine (18Munoz F. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 2000; : 661-664Google Scholar). Other oxidants, including hypochlorous acid and singlet oxygen also bleach this blue dye (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar, 19Harriram A. Govender V. Jonnalagadda S.B. J. Environ. Sci. Health A. 2003; 38: 1055-1064Google Scholar). However, with ozone but not the other oxidants, an atom of oxygen from water was found to be incorporated into isatin sulfonic acid (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar), as in Scheme 1. As evidence of ozone formation, isotopically labeled oxygen from water was also incorporated into isatin sulfonic acid formed by neutrophils. The implications of neutrophils generating ozone are considerable. Yet a definitive mechanism for production has not been established, and alternative explanations for oxidation of the probes have not been excluded. Therefore, we have reexamined the process whereby neutrophils bleach indigo carmine. We show that superoxide can convert indigo carmine to isatin sulfonic acid and that this reaction can explain indigo carmine bleaching by neutrophils. Materials—Indigo carmine, 4β-phorbol 12-myristate 13-acetate (PMA) 1The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; DTPA, diethylenetriaminepenta-acetic acid; SOD, superoxide dismutase; TBA, tetrabutylammoniumhydrogen sulfate; HPLC, high pressure liquid chromatography. , diethylenetriaminepenta-acetic acid (DTPA), horse heart cytochrome c, bovine liver catalase, bovine erythrocyte Cu/Zn superoxide dismutase (SOD), and buttermilk xanthine oxidase were supplied by Sigma. Isatin sulfonic acid, dl-methionine, and tetrabutylammoniumhydrogen sulfate (TBA) were purchased from Aldrich, British Drug House, and Fluka, respectively. 18O water (95%) was purchased from Cambridge Isotope Laboratories, Inc., Andover, MA. Reaction of Indigo Carmine with Stimulated Neutrophils, Xanthine Oxidase, or Ozone—Neutrophils were isolated from the peripheral blood of healthy donors by centrifugation through Ficoll-Hypaque, dextran sedimentation, and hypotonic lysis of contaminating red cells (20Böyum A. Scand. J. Clin. Lab. Invest. 1968; 21: 77-89Google Scholar). They were incubated at a concentration of 5 × 106 cells/ml in 10 mm phosphate-buffered saline (PBS), pH 7.4, containing 140 mm NaCl with 0.5 mm MgCl2, 1 mm CaCl2, 1 mg/ml glucose, and 30 μm indigo carmine. The neutrophils were warmed to 37 °C for 10 min prior to stimulation with 100 ng/ml of PMA. After an additional 20 min, they were pelleted, and supernatants were assayed by HPLC to determine the extent of oxidation of indigo carmine and identify the products of oxidation. Indigo carmine was also bleached with 46 milliunits/ml of xanthine oxidase and 10 mm acetaldehyde in phosphate buffer at pH 7.4. Solutions contained 20 μg/ml of catalase and 100 μm DTPA to block reactions of hydrogen peroxide and hydroxyl radical. The loss of indigo carmine was monitored by following the decline in absorbance at 610 nm and by HPLC. The rate of production of superoxide by xanthine oxidase was determined by measuring cytochrome c reduction over the first minute of reaction (21Fridovich I. Greenwald R.A. Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL1985: 213-215Google Scholar). Ozonolysis was carried out by bubbling ozone through a solution of 680 μm indigo carmine in phosphate buffer until approximately one-third of the indigo carmine had decolorized. Continued ozonolysis resulted in complete loss of isatin sulfonic acid because of further reactions. Ozone was generated by passing molecular oxygen through an OL100/DS ozone generator from Yanco Industries Ltd, Burton, BC, Canada. HPLC Analysis of Indigo Carmine Reactions—Indigo carmine was separated from isatin sulfonic acid using a Waters 2690 HPLC and monitored using a Waters 996 diode array detector. Samples (50 μl) from neutrophil supernatants or the xanthine oxidase system were injected on to a Phenomonex Luna 5 μm C18 reverse-phase column (250 × 4.6 mm) and eluted isocratically with 70% 50 mm phosphate buffer (pH 7.2) containing 10 mm TBA and 30% acetonitrile. The flow rate was 0.8 ml/min. Retention times of indigo carmine and isatin sulfonic acid were determined using authentic standards. Mass Spectral Analysis—Reactions of indigo carmine with xanthine oxidase and ozone were carried out in buffers containing at least 83% H218O as well as in normal water. Isatin sulfonic acid generated by these systems was analyzed by direct injection of 40 μl of sample into a stream of acetonitrile that flowed into a Micromass LCT time-of-flight mass spectrometer with an electrospray ion source. Products were analyzed with negative ionization in the m/z range from 70 to 2500 mass units. Direct injection was necessary because initial separation by HPLC resulted in the exchange of oxygen atoms in isotopically labeled isatin sulfonic acid with water in the eluant. Neutrophils were stimulated in the presence of indigo carmine under conditions similar to those reported by Wentworth et al. (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar). In agreement with their results, we found that indigo carmine was bleached with concomitant production of isatin sulfonic acid (Fig. 1). In 20 min, there was a loss of about 25 μm indigo carmine and an accumulation of a similar concentration of isatin sulfonic acid. To establish what product of neutrophils promoted formation of isatin sulfonic acid, cells were stimulated in the presence of scavengers of reactive oxygen species (Fig. 1). SOD, which scavenges superoxide, completely blocked the loss of indigo carmine and the production of isatin sulfonic acid. Methionine, which reacts rapidly with ozone (22Kanofsky J.R. Sima P.D. Arch. Biochem. Biophys. 1995; 316: 52-62Google Scholar), singlet oxygen (23Michaeli A. Feitelson J. Photochem. Photobiol. 1994; 59: 284-289Google Scholar), and hypochlorous acid (24Peskin A.V. Winterbourn C.C. Free Radic. Biol. Med. 2001; 30: 572-579Google Scholar) had no effect on either the loss of indigo carmine or the formation of isatin sulfonic acid. Catalase, which degrades hydrogen peroxide, and azide, an inhibitor of myeloperoxidase, were also without effect. From these results, it is apparent that superoxide was responsible for converting indigo carmine to isatin sulfonic acid. From the inhibitor studies, no contribution by ozone, hydrogen peroxide, singlet oxygen, or hypochlorous acid was evident. To test whether superoxide can react directly with indigo carmine, we generated it using xanthine oxidase and acetaldehyde. The rate of superoxide generation was chosen to match that of neutrophils under the conditions described in Fig. 1. Catalase was included to prevent reactions of hydrogen peroxide and the metal chelating agent DTPA from stopping production of hydroxyl radical. The xanthine oxidase system slowly bleached indigo carmine (Fig. 2A). Isatin sulfonic acid was the major product formed, as identified by its HPLC retention time (Fig. 2B) and its UV absorbance spectrum (λmax = 245 and 298 nm). Superoxide dismutase prevented indigo carmine loss (Fig. 2A) and inhibited isatin sulfonic acid production by 96% (Fig. 2, B and C). The yield of isatin sulfonic acid (9.2 μm) was 31% of the theoretical maximum, if one molecule of indigo carmine gives two molecules of isatin sulfonic acid. This yield is similar to what was obtained with neutrophils (Fig. 1). The maximum concentration of superoxide that could have been generated during the 10 min of the reaction was 290 μm. It could have been less because of oxygen depletion in the buffer but, regardless, less than 5% of the superoxide reacted to produce isatin sulfonic acid. These results indicate that superoxide reacts slowly with indigo carmine and converts it to isatin sulfonic acid. The neutrophil oxidant responsible for bleaching indigo carmine was found to react in an analogous way to authentic ozone by incorporating one atom of oxygen from water into isatin sulfonic acid (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar). To determine whether this also occurs with superoxide, indigo carmine was reacted with either the xanthine oxidase system or ozone in H218O. Initial studies were performed in normal water to establish that both systems produce isatin sulfonic acid with the predicted m/z of 226 mass units when analyzed in the negative ion mode (Fig. 3, B and D). In H218O, isatin sulfonic acid would be expected to be present predominantly with m/z of 228 because of reversible exchange of its carbonyl oxygen with water. Incorporation of 18O into the amide carbonyl because of involvement of water in the oxidation process (see Scheme 1) would give an additional product with m/z of 230. When indigo carmine was reacted with superoxide (Fig. 3A) or subjected to ozonolysis (Fig. 3C) in H218O, mass analysis revealed products with m/z ratios of 228 and 230 in equal abundance. These results demonstrate that superoxide and ozone leave the same chemical signature when they react with indigo carmine. Much of the evidence for the proposal that neutrophils produce ozone comes from studies with indigo carmine (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar, 6Babior B.M. Takeuchi C. Ruedi J. Gutierrez A. Wentworth P.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3031-3034Google Scholar). We have investigated further the conversion of indigo carmine to isatin sulfonic acid by neutrophils and present strong evidence that superoxide is responsible for the reaction. We found that it was completely inhibited by superoxide dismutase but unaffected by agents that could either block production of ozone or scavenge it directly. Furthermore, superoxide generated by xanthine oxidase also reacted with indigo carmine to give a similar yield of isatin sulfonic acid as with neutrophils and the same oxygen incorporation from water as with ozone. Consequently, the bleaching of indigo carmine by neutrophils cannot be used to support the proposal that these cells produce ozone. Superoxide was discounted previously as the species responsible for indigo carmine bleaching by neutrophils because a 10 mm bolus addition of potassium superoxide did not produce isatin sulfonic acid (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar). We investigated this reaction and also found that, although the indigo carmine was completely bleached, isatin sulfonic acid was not detectable (results not shown). However, a bolus of potassium superoxide would disappear by dismutation with a half-life of less than a second at neutral pH, and formation of isatin sulfonic acid may be inefficient under these conditions. By using xanthine oxidase to mimic the prolonged generation of superoxide by neutrophils over a longer time, oxidation of indigo carmine was analogous to that with neutrophils. The mechanism initially proposed for bleaching of indigo carmine by neutrophils involves antibody-catalyzed oxidation of water by singlet oxygen. However, as discussed in the introductory paragraphs of this paper, it is debatable whether there is sufficient antibody bound to the cells or singlet oxygen produced to support this mechanism. A direct reaction of superoxide is sufficient to explain isatin sulfonic acid formation without requiring either antibodies or singlet oxygen. A possible mechanism for the reaction with superoxide is given in Scheme 2. It involves one-electron reduction of indigo carmine to a radical that reacts with another superoxide to form an organic peroxide. The peroxide could then break down via hydrolysis and liberate isatin sulfonic acid as well as dioxindole sulfonate. The dioxindole sulfonate would contain an atom of oxygen originally from water and would auto-oxidize to isatin sulfonic acid. In support of this mechanism, indigo carmine readily undergoes two-electron reduction to its leuco-form (E′o = -125 mV; Refs. 25Clark W.M. Oxidation-Reduction Potentials of Organic Systems. Waverly Press, Baltimore1960Google Scholar, 26Cook A.G. Tolliver R.M. Williams J.E. J. Chem. Ed. 2003; 71: 160-161Google Scholar). One-electron reduction of indigo carmine (E′o = -247 mV; Ref. 27Preisler P.W. Hill E.S. Loeffel R.G. Shaffer P.A. J. Am. Chem. Soc. 1959; 81: 1991-1995Google Scholar) by superoxide (E′o = -330 mV; Ref. 28Ilan Y.A. Czapski G. Meisel D. Biochim. Biophys. Acta. 1976; 430: 209-225Google Scholar) is thermodynamically likely and is analogous to the reduction of quinones to semiquinones by superoxide (29Brunmark A. Cadenas E. Free Radic. Biol. Med. 1989; 7: 435-477Google Scholar). Based on the one- and two-electron reduction potentials for indigo carmine, the one-electron reduction potential for its radical anion should be about 0 mV. Hence, it should react even more favorably with a second molecule of superoxide. Formation of the organic peroxide is akin to reactions of phenoxyl radicals with superoxide (30Winterbourn C.C. Kettle A.J. Biochem. Biophys. Res. Commun. 2003; 305: 729-736Google Scholar), which occur at almost diffusioncontrolled rates (31d'Allesandro N. Bianchi G. Fang X. Jin F. Schuchmann H.-P. von Sonntag C. J. Chem. Soc. Perkin Trans. II. 2000; : 1862-1867Google Scholar). Wentworth et al. (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar, 6Babior B.M. Takeuchi C. Ruedi J. Gutierrez A. Wentworth P.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3031-3034Google Scholar) also showed that both ozone and neutrophils converted vinylbenzoate to 4-carboxybenzaldehyde and used this as supporting evidence for ozone production by neutrophils. However, it is well established that myeloperoxidase oxidizes styrene derivatives such as vinylbenzoate to benzaldehydes (32Tuynman A. Spelberg J.L. Kooter I.M. Schoemaker H.E. Wever R. J. Biol. Chem. 2000; 275: 3025-3030Google Scholar). Therefore, an alternative explanation for the production of 4-carboxybenzaldehyde by neutrophils is that it occurred by a myeloperoxidase-dependent process. Our findings have ramifications for other biological systems where ozone production is proposed. Much of the evidence that ozone is formed when antibodies catalyze oxidation of water by singlet oxygen comes from studies of indigo carmine oxidation (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar). From the work presented here, the species responsible could be either ozone or superoxide. Also, formation of isatin sulfonic acid in the inflammatory Arthus reaction (4Wentworth P.J. McDunn J.E. Wentworth A.D. Takeuchi C. Nieva J. Jones T. Bautista C. Ruedi J.M. Gutierrez A. Janda K.D. Babior B.M. Eschenmoser A. Lerner R.A. Science. 2002; 298: 2195-2199Google Scholar) and in atherosclerotic plaques (5Wentworth P.J. Nieva J. Takeuchi C. Galve R. Wentworth A.D. Dilley R.B. DeLaria G.A. Saven A. Babior B.M. Janda K.D. Eschenmoser A. Lerner R.A. Science. 2003; 302: 1053-1056Google Scholar) may also involve superoxide, which is known to be produced in these situations (33Grisham M.B. Trends Pharmacol. Sci. 2000; 21: 119-120Google Scholar). More direct and specific methods for detection of ozone will be needed to establish whether this gas is produced in biological systems." @default.
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• W1976760968 title "Superoxide Converts Indigo Carmine to Isatin Sulfonic Acid" @default.
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