Detection and quantitation of hydroxyl radicals in post-ischemic rat kidney
Abstract
Quantitation of hydroxyl (HO*) radicals in post-ischemic tissues is an important step to test the hypothesis that these radicals, generated by the superoxide-driven driven Fenton reaction, play a major role in the inititation of reperfusion injury. The development of quantitative methods, to complement existing qualitative methods, would allow confirmation of the free radical hypothesis explaining reperfusion injury. This thesis describes the development, validation and use of a simple, quantitative method for measuring hydroxyl radicals in ischemic and reoxygenated rat kidney. Hydroxyl radicals are "trapped" with dimethyl sulfoxide (DMSO) and the accumulation of the specific product of that trapping reaction, methane sulfinic acid (MSA), is measured with a novel spectrophotometric assay. The assay is based on the detection of a yellow diazosulfone, formed by the reaction between the sulfinic acid and a diazonium salt. The trapping reaction and assay were found efficient enough to recover close to the theoretical yield HO* formed by known radical-generating systems. Three major modifications to the assay were made to improve specificity and sensitivity for applications in biological samples, and the assay was applied to DMSO pretreated rat kidneys, subjected to a cycle of ischemia and reoxygenation in short term organ culture. Hydroxyl radical formation in postischemic rat kidney between 1 and 30 nmol/gram was considered biologically relevant. Rat kidneys subjected to a cycle of ischemia and reoxygenation produced an average of 7 nmol HO* per gram; this level of HO* formation was not statistically different from nonischemic or from ischemic, but non-reoxygenated control tissues. A large postischemic "burst" of HO* radicals was not observed, because kidney homogenates, even when diluted 1000-fold, were shown to quench HO* formation, by the superoxide-driven Fenton reaction.
Degree
Ph.D.
Advisors
Babbs, Purdue University.
Subject Area
Anatomy & physiology|Animals
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.