Interference of reducing agents with the hematoxylin assay for determination of cupric chelation

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Emily Zsáková Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of diploma thesis: Interference of reducing agents with the hematoxylin assay for determination of cupric chelation Copper is an essential trace element of living organisms. Disorders of its homeostasis affect various pathological conditions. In excessive amounts, it is toxic due to its ability to form free radicals. Vitamin C, trolox (a water soluble form of vitamin E), glutathione and hydroxylamine possess reducing properties that protect our body from oxidative tissue damage. Oxidized glutathione (GSSG) was also tested for comparison. The object of study was to test the chelating ability of these substances. A simple and fast spectrophotometric method was used. The combination of reducing properties of copper and chelating agents could lead to an improvement in chelation therapy either in heavy metal poisoning, treatment of Wilson's disease, or other diseases. We found that all substances were chelators/copper-chelating agents in vitro in a slightly competitive environment, using hematoxylin as an indicator. However, by using the bathocuproine assays to verify chelating properties, none of the substances..