Antioxidant inhibition of oxygen radicals for measurement of total antioxidant capacity in chemical and biological samples

Simon Ching

Research output: ThesisDoctoral Thesis

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A new method has been developed to measure the total antioxidant capacity of chemicals, biological samples such as serum, and everyday substances such as vitamins, tea, coffee, wine, spices, fruits, and vegetables to address important unresolved concerns arising from intervention trials and clinical studies of the beneficial protective effect of antioxidants versus the damaging effect of free radicals and reactive oxygen species in oxidative stress in vivo. A review of previous attempts to resolve these concerns have shown to be hindered by a lack of methods which take into account the two parameters of measurement for assessing total antioxidant capacity: firstly, the degree of inhibition and secondly, the duration of inhibition by antioxidants. In addition, existing fluorescence methods with these fundamentals still require either above ambient temperature incubation, reaction pre-heating and/or separate assays for testing hydrophilic and hydrophobic antioxidant samples. This high throughput "antioxidant inhibition of oxygen radicals" (AIOR) method is performed at ambient temperature and is applicable to samples either in aqueous solution or common organic solvents. The method has good linearity, within- and between-run precision and recovery. The AIOR method uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule uroporphyrin I, which is delayed by the presence of antioxidants. The area under the fluorescence curve is measured by a fluorescence spectrophotometer in a 96-well microplate format with total antioxidant capacity results expressed in mmol/L Trolox equivalents. Many of the concerns associated with the measurement of total antioxidant capacity have been overcome and AIOR has been applied to measure total antioxidant capacity of chemicals and biological samples such as serum. In addition, the kinetics and the reaction mechanism of the AIOR reaction have been studied using UV-visible and fluorescence spectrophotometry, high performance liquid chromatography (HPLC), liquid chromatography mass spectroscopy (LC-MS) and electron spin resonance (ESR) analysis. The reaction between the indicator molecule uroporphyrin I and the alkoxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be first order kinetics with a mean rate constant (k) of 0.0254. A mechanism for the reaction and the breakdown by-products of the reaction is proposed based on the results from these experiments.
Original languageEnglish
QualificationDoctor of Philosophy
Publication statusUnpublished - 2007


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