Assay methods hydroperoxide formation

To learn about the real effects of antioxidants, it is therefore important to obtain specific chemical information about which products of lipid oxidation are inhibited. Several specific assays are needed to elucidate how lipid oxidation products act in the complex multi-step mechanism of lipid oxidative deterioration of foods. The results of several complementary methods are required to determine lipid oxidation products formed at different stages of the free radical chain. Since antioxidants show different activities toward hydroperoxide formation and decomposition, it is important that more than one method be used to monitor Upid oxidation. 

Hydroperoxides cause oxidation of Fe(II) ions to Fe(III) in acid solution. In a modification of the FOX assay (Section III.B.2.a) the oxidized ions form a blue-purple 1 1 complex with Xylenol Orange (71) that can be measured at 560 nm"" """. However, it has been found that formation of a 2 1 complex is also possible and some corrections need be made to obtain a good correlation between this method and POV obtained by the AOCS official method"" . A modification of the FOX method proposed for determination of POV in beef, chicken, fish, butter and vegetable products correlates well with standard iodometric and FOX assays"". ... 

Ferric Ion Complexes Other chemical methods based on the oxidation of ferrous ion (Fe ) to ferric ion (Fe ) in an acidic medium and the formation of iron complexes have also been widely accepted. These methods spectrophotometri-cally measure the abihty of lipid hydroperoxides to oxidize ferrous ions to ferric ions, which are complexed by either thiocyanate or xylenol orange (23, 28, 29). Ferric thiocyanate is a red-violet complex that shows strong absorption at 500-510 nm (8). The method of determining PV by coloremetric detection of ferric thiocyanate is simple, reproducible, and more sensitive than the standard iodometric assay, and has been used to measure hpid oxidation in milk products, fats, oils, and liposomes (8, 23). 

Hydroperoxide lyase assay. Hydroperoxide lyase was determined by measurement of the formation of hexanal from 13-hydroperoxlde of linoleic acid at pH 6.3.(1 ) The substrate (6 pmol) dissolved In diethyl ether was pipetted Into a 50-ml flask and the solvent was evaporated 1 vacuo. Then, 10 ml of chloroplast suspension or leaf homogenate was added. The mixture was sealed In the flask with a rubber stopper and Incubated at 35°C for 10 mLn. The Cg-aldehydes formed were measured by the headspace method with GLC. 

---