Kettle Anthony J, Clark Bruce M, Winterbourn Christine C
Free Radical Research, Department of Pathology, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand.
J Biol Chem. 2004 Apr 30;279(18):18521-5. doi: 10.1074/jbc.M400334200. Epub 2004 Feb 20.
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 H(2)(18)O, the proportion of (18)O 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.
最近，有人提出中性粒细胞会产生臭氧（温特沃思，P.J.，麦克邓恩，J.E.，温特沃思，A.D.，竹内，C.，涅瓦，J.，琼斯，T.，包蒂斯塔，C.，鲁迪，J.M.，古铁雷斯，A.，詹达，K.D.，巴比奥尔，B.M.，埃申莫泽，A.，和勒纳，R.A.（2002年）《科学》298卷，2195 - 2199页；巴比奥尔，B.M.，竹内，C.，鲁迪，J.，古铁雷斯，A.，和温特沃思，P.J.（2003年）《美国国家科学院院刊》100卷，3031 - 3034页）。该提议的证据主要基于臭氧与靛蓝胭脂红反应生成异吲哚酮磺酸的化学过程。在本研究中，我们检验了此反应的特异性，以及它是否可作为中性粒细胞产生臭氧的确凿证据。受刺激的中性粒细胞促使靛蓝胭脂红消失并形成异吲哚酮磺酸，该反应被超氧化物歧化酶完全抑制。甲硫氨酸可清除臭氧、单线态氧和次氯酸，对该反应无影响。过氧化氢酶或叠氮化物也分别对该反应无影响，它们可清除过氧化氢并抑制髓过氧化物酶。从这些结果明显看出，超氧化物是导致靛蓝胭脂红褪色的原因。用黄嘌呤氧化酶和乙醛产生的超氧化物也能将靛蓝胭脂红转化为异吲哚酮磺酸，该反应被超氧化物歧化酶完全抑制且不受过氧化氢酶影响。当黄嘌呤氧化酶反应在H₂¹⁸O中进行时，掺入异吲哚酮磺酸中的¹⁸O比例与臭氧反应时相同。因此，就异吲哚酮磺酸的形成而言，臭氧和超氧化物与靛蓝胭脂红的反应无法区分。我们得出结论，靛蓝胭脂红的褪色不能用于证明中性粒细胞产生了臭氧。在其他生物学过程中使用靛蓝胭脂红来暗示臭氧存在的研究也应谨慎解读。
