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Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils
S. Mueller,
J. Arnhold,
Corresponding Author
S. Mueller
Institute of Medical Physics and Biophysics, School of Medicine, University of Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany
Department of Internal Medicine IV, University of Heidelberg, Bergheimer Strasse 58, 69115 Heidelberg, GermanySearch for more papers by this authorJ. Arnhold
Institute of Medical Physics and Biophysics, School of Medicine, University of Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany
Search for more papers by this authorS. Mueller,
J. Arnhold,
Corresponding Author
S. Mueller
Institute of Medical Physics and Biophysics, School of Medicine, University of Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany
Department of Internal Medicine IV, University of Heidelberg, Bergheimer Strasse 58, 69115 Heidelberg, GermanySearch for more papers by this authorJ. Arnhold
Institute of Medical Physics and Biophysics, School of Medicine, University of Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany
Search for more papers by this authorAbstract
A fast and sensitive chemiluminescence assay for the determination of H2O2 in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide.
Changes in H2O2 concentration in PMA-stimulated neutrophils were followed by injection of NaOCI to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H2O2 without influence of H2O2 decomposition by cellular enzymes or newly produced H2O2 due to dismutation of superoxide anion radicals. Concentrations of H2O2 were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase. Changes in H2O2 concentration upon stimulation could be observed at 3000 cell/mL.
References
- 1 Allen RC, Phagocyte oxygenation activities: quantitative analysis based on luminescence. In: J Schölmerich, R Andreesen, A Kapp, M Ernst, WG Woods, editors. Bioluminescence and chemiluminescence. Chichester: John Wiley, 1987; 13–22.
- 2 Fantone JC, Ward, PA, Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Pathol 1982; 107: 397–418.
- 3
Rossi F,
The O
-forming NADPH oxidase of the phagocytes: Nature, mechanism of activation and function.
Biochim Biophys Acta
1986;
853: 65–89.
- 4 Clifford DP, Repine JE, Measurement of oxidizing radicals by polymorphonuclear leukocytes. Methods Enzymol 1984; 105: 393–8.
- 5 Fridovich I, O2 radicals, hydrogen peroxide, and O2 toxicity. In: WA Pryor, editor. Free radicals in biology, vol. 1. New York: Academic Press, 1976; 272–308.
- 6 Halliwell B, Gutteridge JMC, Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol 1990; 186: 1–85.
- 7 Kanofsky JR, Singlet oxygen production by biological systems. Chem Biol Interact 1989; 70: 1–28.
- 8 Radi R, Cosgrove TP, Beckman JS, Freeman BA, Peroxynitrite-induced luminol chemiluminescence. Biochem J 1993; 290: 51–7.
- 9 Jaeschke H, Reactive oxygen and ischemia/reperfusion injury of the liver. Chem Biol Interact 1991; 79: 115–36.
- 10 Sussman MS, Bulkley GB, Oxygen derived free radicals in reperfusion injury. Methods Enzymol 1990; 186: 711–9.
- 11 De Sole P, Polymorphonuclear chemiluminescence: some clinical applications. J Biolumin Chemilumin 1989; 4: 251–62.
- 12 Makino R, Tanaka T, Iizuka T, Ishimura Y, Kanegasaki S, Stoichiometric conversion of oxygen to superoxide anion during the respiratory burst in neutrophils. Direct evidence by a new method for measurement of superoxide anion with diacetyldeuteroheme-substituted horseradish peroxidase. J Biol Chem 1986; 261: 11444–7.
- 13 Hyslop PA, Sklar LA, A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes. Anal Biochem 1984; 141: 280–6.
- 14 Corbett JT, The scopoletin assay for hydrogen peroxide. A review and a better method. J Biochem Biophys Meth 1989; 18: 297–309.
- 15 Seitz WR, Chemiluminescence detection of enzymatically generated peroxide. Methods Enzymol 1978; 57: 445–62.
- 16 Roswell DF, White EH, The chemiluminescence of luminol and related hydrazides. Methods Enzymol 1978; 57: 409–23.
- 17 Wymann MP, von Tscharner V, Deraulean DA, Baggiolini M, Chemiluminescence detection of H2O2 produced by human neutrophils during the respiratory burst. Anal Biochem 1987; 165: 371–8.
- 18 Lock R, Johansson A, Orselius K, Dahlgren C, Analysis of horseradish peroxidase-amplified chemiluminescence produced by human neutrophils reveals a role for the superoxide anion in the light emitting reaction. Anal Biochem 1988; 173: 450–5.
- 19 Dahlgren C, Polymorphonuclear leukocyte chemiluminescence induced by formylmethionyl-leucyl-phenylalanine and phorbol myristate acetate: effects of catalase and superoxide dismutase. Agents Actions 1987; 21: 104–12.
- 20 Misra HP, Squatrito PM, The role of superoxide anion in peroxidase-catalysed chemiluminescence of luminol. Arch Biochem Biophys 1982; 215: 59–65.
- 21 Merényi G, Lind J, Eriksen TE, Luminol chemiluminescence: chemistry, excitation, emitter. J Biolumin Chemilumin 1990; 5: 53–6.
- 22 Eriksen TE, Lind J, Merényi G. Oxidation of luminol by chlorine dioxide: formation of 5-amino-phthalazine-1,4-dione. J Chem Soc Faraday Trans 1981; 77: 2125–35.
- 23 Lind J, Merényi G, Eriksen TE, Chemiluminescence mechanism of cyclic hydrazides such as luminol in aqueous solutions. J Am Chem Soc 1983; 105: 7655–61.
- 24 Albrecht HO, Über die Chemiluminescenz des Aminophthalsäurehydrazids. Z physikal Chem 1928; 136: 321–330.
- 25 Isacsson K, Wettermark G, The determination of inorganic chlorine compounds by chemiluminescence reactions. Anal Chim Acta 1976; 83: 227–39.
- 26 Brestel EP, Co-oxidation of luminol by hypochlorite and hydrogen peroxide: implications for neutrophil chemiluminescence. Biochem Biophys Res Commun 1985; 126: 482–8.
- 27 Arnhold J, Mueller S, Arnold K, Grimm E, Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide. J Biolumin Chemilumin 1991; 6: 189–92.
- 28 Arnhold J, Mueller S, Arnold K, Sonntag K, Mechanisms of inhibition of chemiluminescence in the oxidation of luminol by sodium hypochlorite. J Biolumin Chemilumin 1993; 8: 307–13.
- 29 Mueller S, Arnhold J, Verfahren zur Bestimmung von Wasserstoffperoxid. 8. 4. 1993 De 43 12 097 A1 (patent).
- 30 Morris JC. The acid ionization constant of HOCl from 5 to 35°C. J Phys Chem 1966; 70: 3798–806.
- 31 Beers RF, Sizer IW, A spectrometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 1952; 195: 133–40.
- 32 Aebi H, Catalase in vitro. Methods Enzymol 1984; 105: 121–6.
- 33 Klebanoff SJ, Myeloperoxidase: contribution to the microbicidal activity of intact leukocytes. Science 1970; 169: 1095–7.
- 34 Dahlgren C, Follin P, Johansson A, Lock R, Orselius K, Localization of the luminol-dependent chemiluminescence reaction in human granulocytes. J Biolumin Chemilumin 1989; 4: 263–6.
- 35 Test ST, Weiss SJ, Quantitative and temporal characterization of the extracellular H2O2 pool generated by human neutrophils. J Biol Chem 1984; 259: 399–405.
- 36 Winterbourn CC, Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarities of the oxidant hypochlorite. Biochim Biophys Acta 1985; 840: 204–10.
- 37 Arnhold J, Hammerschmidt D, Wagner M, Mueller S, Arnold K, Grimm E, On the action of hypochlorite on human serum albumin. Biomed Biochim Acta 1990; 49: 991–7.
- 38 Arnhold J, Hammerschmidt S, Arnold K, Role of functional groups of human serum albumin in scavenging of NaOCl and neutrophil derived hypochlorous acid. Biochem Biophys Acta 1991; 1097: 145–51.
- 39 Merényi G, Lind J, Eriksen TE, Nucleophilic addition to diazaquinones. Formation and breakdown of tetrahedral intermediates in relation to luminol chemiluminescence. J Am Chem Soc 1986; 108: 7716–26.
- 40 Nahum A, Wood LD, Sznajder JI, Measurement of hydrogen peroxide in plasma and blood. Free Rad Biol Med 1989; 6: 479–84.
- 41 Nurcombe HL, Edwards SW, Role of myeloperoxidase in intracellular and extracellular chemiluminescence of neutrophils. Ann Rheum Dis 1989; 48: 56–62.
- 42 Scott MD, Lubin BH, Kuypers FA, Erythrocyte defense against hydrogen peroxide: preeminent importance of catalase. J Lab Clin Med 1991; 118: 7–16.
- 43 Makino N, Mochizuki Y, Bannai S, Sugita Y, Kinetic studies on the removal of extracellular hydrogen peroxide by cultured fibroblasts. J Biol Chem 1994; 269: 1020–5.
- 44 Halliwell B, Gutteridge JMC, Iron as a biological prooxidant. ISI Atlas Sci Biochem 1988; 1: 48–52.
- 45 DeChatelet LR, Long GD, Shirley PS, Bass DA, Thomas MJ, Henderson W, Cohen MS, Mechanism of luminol-dependent chemiluminescence of human neutrophils. J Immunol 1982; 129: 1589–93.
- 46 Dahlgren C, Stendahl O, Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 1983; 39: 736–41.
