Spontaneous generation of reactive oxygen species in the mixture of cyanide and glycerol.
Reactive oxygen species are involved in tumor promotion or apoptosis. In assaying prooxidant or antioxidant activities, cyanide has been commonly used as an inhibitor of mitochondrial oxidases, peroxidases, or Cu,Zn-superoxide dismutase, which have an influence on intracellular levels of reactive oxygen species. It has also been used to chemically mimic hypoxia. On the other hand, glycerol has been widely used as a stabilizer of various enzymes. In particular, glycerol is required to maintain the enzymatic activities of membrane-bound NAD(P)H oxidases extracted from surrounding phospholipids. Since both cyanide and glycerol are relatively inert, they have been used concomitantly regardless of any mutual interference. In this study, we demonstrate that a mixture of glycerol and cyanide reduced cytochrome c and nitroblue tetrazolium, both of which are superoxide anion indicators. The mixture also enhanced the production of superoxide anion in the presence of redox-cycling compounds. Superoxide production by the mixture was confirmed by electron spin resonance spectra. Moreover, the mixture induced lipid peroxidation and hemolysis in human erythrocytes. These results suggest that cyanide and glycerol should be used carefully in reaction systems used to measure superoxide production or antioxidant activity. However, sucrose and sodium azide in combination do not produce such artifacts and thus may be used as an alternative.
Chun
Yang-Sook
YS
Department of Pharmacology and Cancer Research Institute, Seoul National University, College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799 Korea.
Yeo
Eun-Jin
EJ
Suh
Hwa-Jin
HJ
Park
Jong-Wan
JW
D003486:Cyanides / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D015227:Lipid Peroxidation; D009580:Nitroblue Tetrazolium / Q000737:chemistry; D010269:Paraquat / Q000737:chemistry; D017382:Reactive Oxygen Species; D024483:Vitamin K 3 / Q000737:chemistry
D003486:Cyanides; D017382:Reactive Oxygen Species; D009580:Nitroblue Tetrazolium; D024483:Vitamin K 3; D045304:Cytochromes c; D005990:Glycerol; D010269:Paraquat