Development of assays for biomarkers of oxidative damage to assess the efficacy of fruit-derived antioxidants : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Abstract

The diet is a very important part of maintaining a healthy lifestyle. Increased consumption of fruits and vegetables is one practice postulated to decrease the incidence of diseases such as cancer, cardiovascular disease and other disorders. Although there are a number of possible beneficial compounds in fruit, it is believed that the antioxidant components found in these foods may decrease the oxidative damage that could lead to such diseases. Oxidative damage to cellular proteins, lipids and DNA is considered to result from an increase in the production of free radicals, which overwhelm the body's defence system. This research investigated fruit-derived antioxidants, and developed biomarker assays to measure the potential health benefits they may offer. To determine the in vivo antioxidant efficacy of berry fruit anthocyanins, oxidative damage to proteins, lipids and DNA was measured in rats fed several combinations of natural and synthetic diets. Mild oxidative damage was induced by the inclusion of fish oil in these diets. DNA oxidation was determined by measuring urinary 8-hydroxy-2'-deoxyguanosine using reversed-phase high performance liquid chromatography with electrochemical detection. ELISA and colorimetric techniques were used to measure protein carbonyl content of plasma as a reflection of protein oxidation. Oxidation to lipids was assessed by measuring malondialdehyde, which results from lipid peroxidation. Supplementation with fish oil induced a mild form of dietary oxidative damage, as shown by an increase in lipid and protein oxidation. In most cases the berry fruit extracts had little effect on the level of fish oil-induced oxidative damage, however, boysenberry anthocyanin extract significantly reduced protein oxidation when used in combination with the natural diet. Taken together the results suggest that oxidative damage to biomacromolecules may occur by different pathways of oxidative stress, which selectively target either DNA, protein or lipids at varying levels, and the antioxidant is effective only with selected mechanisms of oxidative damage.