Hydroperoxides are the primary oxygenated products of polyunsaturated fatty acids and were determined spectrophotometrically based on their reaction with an excess of Fe at low pH in the presence of the dye Xylenol Orange. Triphenylphosphine-mediated hydroxide formation was used to authenticate the signal generated by the hydroperoxides. The method readily detected lipid peroxidation in a range of plant tissues including Phaseolus hypocotyls (26±5 nmol·g of fresh weight; mean±S.D.), Alstroemeria floral tissues (sepals, 66±13 nmol·g of fresh weight; petals, 49±6 nmol·g of fresh weight), potato leaves (3344±75 nmol·g of fresh weight), broccoli florets (568±68 nmol·g of fresh weight) and Chlamydomonas cells (602±40 nmol·g of wet weight). Relative to the total fatty acid content of the tissues, the percentage hydroperoxide content was within the range of 0.6-1.7% for all tissue types (photosynthetic and non-photosynthetic) and represents the basal oxidation level of membrane fatty acids in plant cells. Leaves of transgenic potato with the fatty acid hydroperoxide lyase enzyme expressed in the antisense orientation were elevated by 38%, indicating a role for this enzyme in the maintenance of cellular levels of lipid hydroperoxides.
- membrane oxidation
- FOX2, ferrous oxidation of Xylenol Orange (version 2)
- LHPO, lipid hydroperoxide