Oxidative Stress, Glycation and Transition Metals in Diabetic Complications

This thesis discusses the role of transition metal catalysed oxidative reactions in the development of diabetic complications and ageing through in vitro studies. The possible relationship between metal-dependent oxidative stress and non-enzymatic glycosylation (glycation), as well as polyol pathway metabolism, is also discussed. The experimental research reported in this thesis includes the following: 1) A simple and sensitive method for the direct measurement of peroxides is described. The method is based on the principle of the rapid peroxide mediated oxidation of Fe2+ to Fe3+ under acidic conditions. The latter, in the presence of xylenol orange, forms a Fe3+-xylenol orange complex which can be measured spectrophotometrically at 560nm. By pre-treatment with enzymes known to metabolize peroxides, it has been shown that the assay measures hydrogen peroxide (H2O2) and Lipid hydroperoxides specifically. The Ferrous Oxidation/Xylenol orange (FOX) assay has been validated in the study of H2O2 generation during glucose "autoxidation", as well as lipid peroxidation of low density lipoprotein (LDL), phosphatidyl choline liposomes and red blood cell membranes. 2) The interaction between glycation and peroxidation in determining the toxicity of in vitro glucose-modified LDL to a fibroblast cell line is examined. Such modified LDL is cytotoxic, but this is determined primarily by the extent of metal-catalysed peroxidation of the lipoprotein, and that glycation may enhance (by an unknown mechanism) the cytotoxic effect of oxidized LDL. 3) Some experimental inhibitors (sorbinil, Al-1576 and ONO-2235) of the enzyme aldose reductase (implicated in diabetes mellitus via its ability to catalyse glucose reduction to sorbitol) are potent inhibitors of transition metal-catalysed ascorbate oxidation, and inhibit H2O2 formation by monosaccharide "autoxidation". This inhibition appears to be dependent upon the presence of a spirohydantoin group and/or similar structure. Furthermore, these compounds can also inhibit copper/ascorbate induced cytotoxicity in fibroblasts as judged by thymidine incorporation. It is conceivable that the copper and iron-binding capacity of these compounds may contribute to some of their observed biological effects and may provide a starting point for a new generation of experimental drugs for the treatment of diabetic complications