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J. Biol. Chem., Vol. 279, Issue 18, 18521-18525, April 30, 2004

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Superoxide Converts Indigo Carmine to Isatin Sulfonic Acid

IMPLICATIONS FOR THE HYPOTHESIS THAT NEUTROPHILS PRODUCE OZONE^*

Anthony J. Kettle, Bruce M. Clark¶, and Christine C. Winterbourn

From the Free Radical Research, Department of Pathology, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand and ^¶Department of Chemistry, University of Canterbury, Christchurch, New Zealand

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.

Received for publication, January 13, 2004 , and in revised form, February 20, 2004.

^* This work was supported by the Health Research Council of New Zealand. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology, Christchurch School of Medicine and Health Sciences, P.O. Box 4345, Christchurch, New Zealand. Tel.: 643-3640-577; Fax: 643-3641-083; E-mail: tony.kettle{at}chmeds.ac.nz.

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