Measurement of phosphatidylcholine hydroperoxides in solution and in intact membranes by the ferric-xylenol orange assay

Formation of a colored complex between ferric iron and xylenol orange (XO) has been used for the determination of hydroperoxides (FOX method). Original or modified FOX methods were performed on aqueous or organic solutions consisting of a single phase. However, for lipid peroxides in heterogeneous samples, such as biological materials, much of the lipid is sequestered in a separate phase. Organic solvent extraction of these lipids is often incomplete and may result in additional peroxidation during the extraction procedure. In this study, we applied the FOX assay for measurement of the membrane phosphatidylcholine hydroperoxides (PC-OOH) in separated phases. The presence of membranous egg yolk phosphatidylcholine (EYPC) in 60% MeOH shifted the broad peak at 560 nm of Fe³⁺-XO complex to 610 nm with a sharp peak associated with the increased intensity of the absorbance. The shift of the peak is useful to measure the unknown amounts of Fe³⁺ because the uncomplexed XO considerably contributed to the absorbance of the peak at 560 nm but did not affect the absorbance at 610 nm. EYPC was required to form the membranes to shift the peak because the shift occurred in 60% MeOH but did not by the treatment with detergents or in 90% MeOH in which EYPC did not form the membranes. The molar absorption coefficient (ε 610) was 32,700 M^-1 cm^-1, which was about twice the molar absorption co efficient (ε 560) reported. We applied this method to the assay of PC-OOH prepared from EYPC and obtained the molar absorption coefficients (ε 610), which were 79,100 and 115,700 M^-1 cm^-1 in the presence and absence of BHT, respectively. This finding allows the determination of PC-OOH concentration even in chemically complex systems.