Scatchard analysis of methane sulfinic acid production from dimethyl sulfoxide: a method to quantify hydroxyl radical formation in physiologic systems.
A major impediment to the confirmation of free radical mechanisms in pathogenesis is a lack of direct, chemical evidence that oxygen centered free radicals actually arise in living tissues in quantities sufficient to cause serious damage. This investigation was conducted to validate the use of dimethyl sulfoxide (DMSO) as a quantitative molecular probe for the generation of hydroxyl radicals (HO.) under physiologic conditions. Reaction of HO. with DMSO produces methane sulfinic acid (MSA) as a primary product, which can be detected by a simple colorimetric assay. To develop a method for estimating total HO. production, we studied two model systems: the superoxide driven Fenton reaction in vitro, using xanthine oxidase as the source of superoxide, and a computer model of Fenton chemistry. Measured MSA production both in vitro and in the computer model was a predictable function of the concentrations of DMSO and competing scavengers of HO., according to the principle of competition kinetics. Both experimental results and model calculations showed that Scatchard analysis may be used to infer total HO. generation, despite the presence of scavengers other than DMSO, such as mannitol. Thus, methane sulfinic acid production from DMSO holds promise as an easily measured marker for HO. formation in biologic systems pretreated with DMSO, and Scatchard analysis of repeated experiments with varying DMSO concentrations can yield an estimate of total HO. generation.
Babbs
C F
CF
Hillenbrand Biomedical Engineering Center, Purdue University, West Lafayette, IN 47907.
Griffin
D W
DW
D003124:Colorimetry; D004121:Dimethyl Sulfoxide / Q000378:metabolism*; D005609:Free Radicals; D006900:Hydroxylation; D007700:Kinetics; D008954:Models, Biological; D013056:Spectrophotometry, Ultraviolet; D013441:Sulfinic Acids / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism
D005609:Free Radicals; D013441:Sulfinic Acids; C053310:methanesulfinic acid; D014969:Xanthine Oxidase; D004121:Dimethyl Sulfoxide