Measurements of the antioxidant status of biological materials have a number of important applications, such as the evaluation of natural and synthetic pharmaceutical preparations, isolation of novel antioxidants from biological sources and early diagnosis of pathologies suspected to be caused by an accumulation of reactive oxygen species (ROS). However, attempts to devise reliable in vitro assays for the determination of total antioxidant activities of biological samples is rendered complicated by the heterogeneity of the ROS, their complex interactions, the variety and varied susceptibility of biological targets that they attack and the differences in the mechanisms of actions of the antioxidants themselves. Based on the results that we have obtained in Phase I of this project, we propose to undertake the following work in Phase II. A) We will develop a large battery of assay systems which will take into account all of the critical factors that may influence the assessment of antioxidant activity, for example: a) the types of ROS; b) the systems for the generation of these ROS; c) the nature of the molecular targets and d) the methods used for the measurement of the effects of ROS. B) We will optimize two separate series of assay systems which differ from each other on the nature and accessibility of the target molecule a) assays in which the target molecule is present as an integral part of a cell structure in a native environment (intact-cell systems). In this manner, we propose to examine critically 70 different cell-free assays and 64 different intact-cell assays, in which 5 types of ROS produced from 8 different ROS generating systems will be examined. As target s of these ROS, we will employ six different chemical agents, four enzyme proteins an to polyunsaturated fatty acids. Neurons and astrocytes, their mitochondria, membranes and mitochondrial DNA will serve as target of ROS in the intact-cell assays. C) Using these systems, we will test, individually and in various combinations two different groups of antioxidants. The first group consists of 3 hydrophilic and 5 lipophilic compounds which can be obtained c commercially in pure form. The second group consists of concentrates of plants which are known to contain various hydrophilic and lipophilic antioxidants of medicine value in unknown proportions. D) Based on the results of these cell-free and intact-cell systems, we will select a minimum number of operationally simple assays that will permit us to assign an index of total antioxidant activity to test samples.
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