Using a modified ferrous oxidation-xylenol orange (FOX) assay for detection of lipid hydroperoxides in plant tissue

The ferrous oxidation-xylenol orange (FOX) assay was adapted for quantifying lipid hydroperoxides (LOOHs) in plant extracts. Excised pieces of several fruit and vegetable species were exposed to 83 kJ m(-2) day(-1) of biologically effective ultraviolet B irradiance (UV-B(BE)) for 10-12 days to induce cellular oxidation. The LOOH and thiobarbituric acid reactive substance (TBARS) concentrations of these plant tissues were assessed with the FOX and iodometric assays for the former and a modified TBARS assay for the latter. There was generally good agreement between the FOX and iodometric methods both prior to and following the UV exposure. However, the iodometric assay appeared to have some difficulty in consistently quantifying lower LOOH levels (<11 microM), whereas the FOX assay measured LOOH concentrations as low as 5 microM. All tissues exhibited UV-induced increases in TBARS, indicating a marked degree of cellular oxidation in the exposed tissue segments. Compared with the iodometric assay, the FOX method consistently generated less variable LOOH values. The presence of authentic linoleic acid-OOHs in spiked avocado and spinach samples (11 microM) was identified with liquid chromatography-mass spectrometry techniques, which validated corresponding FOX assay results. The FOX method is inexpensive, is not sensitive to ambient O2 or light levels, and can rapidly generate LOOH measurements. The physiological value of the FOX assay resides in its ability to measure initial rather than more advanced fatty acid oxidation; hence, early membrane-associated stress events in plant tissue can be detected.