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Quantitation of the hydroxyl radical adducts of salicylic acid by micellar electrokinetic capillary chromatography: oxidizing species formed by a Fenton reaction

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Abstract

There has been controversy concerning the products formed by a Fenton reaction. We determined the hydroxyl radical (•OH) generated in a Fenton reaction system with no iron chelator using micellar electrokinetic capillary chromatography (MECC). The hydroxyl radical generated in this Fenton system attacked salicylic acid to produce major products of 2,3- and 2,5-dihydroxybenzoic acid (DHB), 2,3-DHB being prominent. Hydroxyl radical scavengers, such as mannitol, ethanol, thiourea and a ferric chelator, Desferal, significantly diminished the peaks for DHBs, showing production of •OH. We compared the MECC method with the electron paramagnetic resonance (EPR) spin trapping technique. The quantity of DHBs obtained by MECC increased dose-dependently up to 1 μM Fe2+ at a fixed concentration of H2O2, whereas that of the spin adduct by EPR showed a bell-shaped curve. This quantitation of •OH adducts by MECC supports the proposal that the oxidizing species formed by a Fenton reaction with no chelator is •OH. The EPR spin trapping method appears to be erroneous, particularly when iron is present at a higher concentration than hydrogen peroxide. The application of this method to the paraquat effect in vitro is demonstrated, and the possibility for analysis of •OH in vivo is also discussed.

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References

  • Bors W, Michel C, Saran M (1979) On the nature of biochemically generated hydroxyl radicals. Studies using the bleaching of p-nitrosodiumethylaniline as a direct assay method. Eur J Biochem 95: 621–627

    Article  PubMed  CAS  Google Scholar 

  • Britigan BE, Cohen MS, Rosen GM (1987) Detection of the production of oxygen-centered free radicals by human neutrophils using spin trapping techniques: a critical perspective. J Leukocyte Biol 41: 349–362

    PubMed  CAS  Google Scholar 

  • Buettner GR (1987) Spin trapping: ESR parameters of spin adducts. Free Rad Biol Med 3: 259–303

    Article  PubMed  CAS  Google Scholar 

  • Burkitt MJ (1993) ESR spin trapping studies into the nature of the oxidizing species formed in the Fenton reaction: pitfalls associated with the use of 5,5-dimethyl-1-pyrroline-N-oxide in the detection of the hydroxyl radical. Free Rad Res Commun 18: 43–57

    Article  CAS  Google Scholar 

  • Burkitt MJ, Gilbert BC (1990) Model studies of the iron-catalysed Harber-weiss cycle and the ascorbate-driven Fenton reaction. Free Rad Res Commun 10: 265–280

    Article  CAS  Google Scholar 

  • Cleland LG, Lowthian PJ, Imhoff D, Bochner F, Betts WH, O'Callaghan J (1985) Plasma and synovial fluid gentisate in patients receiving salicylate therapy. J Rheumatol 12: 136–139

    PubMed  CAS  Google Scholar 

  • Das DK, Cordis GA, Rao PS, Liu X, Maity S (1991) High-performance liquid chromatographic detection of hydroxylated benzoic acids as an indirect measure of hydroxyl radical in heart: its possible link with the myocardial reperfusion injury. J Chromatogr 536: 273–282

    Article  PubMed  CAS  Google Scholar 

  • Deby C, Goutier R (1990) New perspectives on the biochemistry of superoxide anion and the efficiency of superoxide dismutases. Biochem Pharmacol 39: 399–405

    Article  PubMed  CAS  Google Scholar 

  • Finkelstein E, Rosen GM, Rauckman EJ (1980a) Spin trapping. Kinetics of the reaction of superoxide and hydroxyl radicals with nitrones. J Am Chem Soc 102: 4994–4999

    Article  CAS  Google Scholar 

  • Finkelstein E, Rosen GM, Rauckman EJ (1980b) Spin trapping of superoxide and hydroxyl radical: practical aspects. Arch Biochem Biophys 200: 1–16

    Article  PubMed  CAS  Google Scholar 

  • Finkelstein E, Rosen GM, Rauckman EJ (1982) Production of hydroxyl radical by decomposition of superoxide spin-trapped adducts. Mol Pharmacol 21: 262–265

    PubMed  CAS  Google Scholar 

  • Floyd RA, Henderson R, Watson JJ, Wong PK (1986) Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCED) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats. Free Rad Biol Med 2: 13–18

    CAS  Google Scholar 

  • Fridovich I (1986) Biological effects of the superoxide radical. Arch Biochem Biophys 247: 1–11

    Article  PubMed  CAS  Google Scholar 

  • Grootveld M, Halliwell B (1986) Aromatic hydroxylation as a potential measure of hydroxyl-radical formation in vivo — identification of hydroxylated derivatives of salicylate in human body fluids. Biochem J 237: 499–504

    PubMed  CAS  Google Scholar 

  • Halliwell B (1978) Superoxide-dependent formation of hydroxyl radicals in the presence of iron chelates. Is it a mechanism for hydroxyl radical production in biochemical systems? FEBS Lett 92: 321–326

    Article  PubMed  CAS  Google Scholar 

  • Halliwell B, Grootveld M (1987) The measurement of free radical reactions in humans — some thoughts for future experimentation. FEBS Lett 213: 9–14

    Article  PubMed  CAS  Google Scholar 

  • Halliwell B, Gutteridge JMC (1985) Free radicals in biology and medicine. Clarendon Press, London.

    Google Scholar 

  • Halliwell B, Gutteridge JMC (1986) Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 246: 501–514

    Article  PubMed  CAS  Google Scholar 

  • Hiller KO, Hodd PL, Willson RL (1983) Anti-inflammatory drugs: protection of a bacterial virus as an in vitro biological measure of free radical activity. Chem Biol Interact 47: 293–305

    Article  PubMed  CAS  Google Scholar 

  • Kaur H, Fagerheim I, Grootveld M, Puppo A, Halliwell B (1988) Aromatic hydroxylation of phenylalanine as an assay for hydroxyl radicals: application to activated human neutrophils and to the heme protein leghemoglobin. Anal Biochem 172: 360–367

    Article  PubMed  CAS  Google Scholar 

  • Kornbrust DJ, Mavis RD (1980) Microsomal lipid peroxidation. I. Characterization of the role of iron and NADPH. Mol Pharmacol 17: 400–407

    PubMed  CAS  Google Scholar 

  • Maskos Z, Rush JD, Koppenol WH (1990) The hydroxylation of the salicylate anion by a Fenton reaction and i-radiolysis: a consideration of the respective mechanisms. Free Rad Biol Med 8: 153–162

    Article  PubMed  CAS  Google Scholar 

  • McCord JM, Day ED (1978) Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett 86: 139–142

    Article  PubMed  CAS  Google Scholar 

  • Montgomery PR, Sitar DS (1981) Increased serum salicylate metabolites with age in patients receiving chronic acetylsalicylic acid therapy. Gerontology 27: 329–333

    Article  PubMed  CAS  Google Scholar 

  • Peters JH, Gordon GR, Kashiwase D, Lown JW, Yen SF, Plambeck JA (1986) Redox activities of antitumor anthracyclines determined by microsomal oxygen consumption and assays for superoxide anion and hydroxyl radical generation. Biochem Pharmacol 35: 1309–1323

    Article  PubMed  CAS  Google Scholar 

  • Pietronigro OB, Jones WB, Kalty K, Demopoulos HB (1977) Interaction of DNA and liposomes as a model for membrane-mediated DNA damage. Nature 267: 78–79

    Article  PubMed  CAS  Google Scholar 

  • Richmond R, Halliwell B, Chauhan J, Darbre A (1981) Superoxide-dependent formation of hydroxyl radicals: detection of hydroxyl radicals by the hydroxylation of aromatic compounds. Anal Biochem 118: 328–335

    Article  PubMed  CAS  Google Scholar 

  • Rumble RH, Roberts MS, Wanwimolruk S (1981) Determination of aspirin and its major metabolites in plasma by high-performance liquid chromatography without solvent extraction. J Chromatogr 225: 252–260

    Article  PubMed  CAS  Google Scholar 

  • Rush JD, Koppenol WH (1986) Oxidizing intermediates in the reaction of ferrous EDTA with hydrogen peroxide. J Biol Chem 261: 6730–6733

    PubMed  CAS  Google Scholar 

  • Rush JD, Koppenol WH (1989) Reactive intermediates formed by the interaction of hydrogen peroxide and ferrous complexes. In: Beaumont PC, Deebble DJ, Parsons BJ, Rice-Evans C (eds) Free radicals, metal ions and biopolymers, Richelieu Press, London, pp 33–44

    Google Scholar 

  • Sakurai K, Ogiso T (1991) Inhibitory effect of glutathione on the generation of hydroxyl radicals in the reaction system of glutathione-alloxan. Chem Pharm Bull 39: 737–742

    CAS  Google Scholar 

  • Steiner MG, Babbs CF (1990) Quantitation of the hydroxyl radical by reaction with dimethyl sulfoxide. Arch Biochem Biophys 278: 478–481

    Article  PubMed  CAS  Google Scholar 

  • Terabe S, Otsuka K, Ando T (1985) Electrokinetic chromatography with micellar solution and open-tubular capillary. Anal Chem 57: 834–841

    Article  CAS  Google Scholar 

  • Tomita M, Okuyama T, Ueki A, Watanabe H, Kawai S (1992) Combined action of paraquat and superoxide on the peroxidation of detergent-dispersed linolenic acid. Biochim Biophys Acta 1128: 174–180

    PubMed  CAS  Google Scholar 

  • Winston GW, Cederbaum AI (1986) Differential effects of the cytochrome P-450/reductase ratio on the oxidation of ethanol and the hydroxyl radical scavenging agent 2-keto-4-thio-methylbutyric acid (KMBA). Biochem Pharmacol 35: 4053–4058

    Article  PubMed  CAS  Google Scholar 

  • Yamazaki I, Piette LH (1990) ESR spin-trapping studies on the reaction of Fe2+ ions with H2O2-reactive species in oxygen toxicity in biology. J Biol Chem 265: 13589–13594

    PubMed  CAS  Google Scholar 

  • Yamazaki I, Piette LH (1991) ESR spin-trapping study on the oxidizing species formed in the reaction of the ferrous ion with hydrogen peroxide. J Am Chem Soc 113: 7588–7593

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Legal Medicine, Kawasaki Medical School, 577 Matsushima, 701-01, Kurashiki, Japan

    Masafumi Tomita & Toshiko Okuyama

  2. Department of Pharmacology, Kawasaki Medical School, 577 Matsushima, 701-01, Kurashiki, Japan

    Satoru Watanabe

  3. Department of Clinical Nutrition, Faculty of medical Professions, Kawasaki University of Medical Welfare, 288 Matsushima, 701-01, Kurashiki, Japan

    Hiroko Watanabe

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  1. Masafumi Tomita
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  2. Toshiko Okuyama
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  3. Satoru Watanabe
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  4. Hiroko Watanabe
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Tomita, M., Okuyama, T., Watanabe, S. et al. Quantitation of the hydroxyl radical adducts of salicylic acid by micellar electrokinetic capillary chromatography: oxidizing species formed by a Fenton reaction. Arch Toxicol 68, 428–433 (1994). https://doi.org/10.1007/s002040050093

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  • DOI: https://doi.org/10.1007/s002040050093

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