Selenium Compounds and Antioxidant Capacity in Bovine Milk. Studies on Glutathione Peroxidase, Bioavailability and Electrochemical Techniques

Abstract: The efficacy and function of antioxidants have been intensively studied in foods and other biological systems in recent decades. This thesis concerns different aspects of the antioxidant properties of bovine milk, with emphasis on measurement of the total antioxidant capacity (TAC), the activity of the selenium-containing glutathione peroxidase (GSHPx), and the bioavailability of selenium from bovine milk in humans. The TAC measurements were based on the scavenging of the cation radical of 2,2?-azinobis(3-ethylenebenzothiazoline-6-sulphonic acid) (the ABTS method), the reduction of the complex of ferric ion and 2,4,6-tripyridyl-s-triazine (the FRAP method), or the amperometric signal registered at a glassy carbon electrode at an oxidation potential of +700 mV (versus AgôAgCl) in a flow-injection assay (the FIAmp method). Milk, whey, high-molecular-weight (HMW, >10 kDa) fraction and low-molecular-weight (LMW, <10 kDa) fraction of whey were all found to have antioxidant capacity. High correlations were found for the TAC values in the LMW fraction as detected by the three methods (R2 > 0.8, P< 0.001). The major antioxidant in the LMW fraction was identified as urate by specific enzyme treatment. Other electrochemical techniques such as cyclic voltammetry (CV) and rotating disk electrode (RDE) linear sweep voltammetry were also employed to characterise the antioxidant capacity in milk. Extracellular glutathione peroxidase (eGSHPx) is one of the antioxidant enzymes in bovine milk, and a coupled enzymatic assay has been optimised for measuring its activity. The activity of GSHPx was monitored during heat treatment of milk and whey. The activity of GHSPx in milk and whey was maintained at 72°C for 2 min, implying that the enzyme was stable at this common pasteurisation temperature used in dairy industry. The activity of purified cellular glutathione peroxidase (cGSHPx) was less stable than that of purified eGSHPx, and both purified enzymes were less stable than the GSHPx activity in milk. Selenium is an integral part of GSHPx, as well as of other selenoproteins with redox enzymatic activities and it is probably also related to the antioxidant status in humans. Therefore, the bioavailability of selenium from bovine milk was studied in humans using the ileostomy model. It was found that the fractional absorption of selenium was significantly higher for milk than for fermented milk, 73.3 (16.1) versus 64.1 (11.2) %, mean (SD), and selenium from bovine milk was thus highly bioavailable to humans.