Measurement of Lipid Hydroperoxides by the Ferric-Xylenol Orange Method (1) : Characteristics of the Ferric-Xylenol Orange/Membrane Phosphatidylcholine Complex

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The ferric-xylenol orange (FOX) method for measurement of hydroperoxides is based on a technique that employs reduction of peroxides in an acidic condition by Fe^<2+> and formation of the colored ferric-xylenol orange (XO/Fe^<3+>) product with a peak at 560nm. The 560nm absorbance peak of XO/Fe^<3+> shifts to a 610nm peak with high absorption intensity in the presence of phosphatidylcholine. This is useful for quantification of peroxides such as phospholipid hydroperoxides. Based on this finding, we recently reported a modified FOX method. We now show by measurements of absorbance, broadening of the electron paramagnetic resonance spectrum, changes in the vesicle size and their zeta potentials, the effects of detergents, and manipulation of the membrane lipid composition that the XO/Fe^<3+>-phosphatidylcholine complex forms only in the presence of intact phosphatidylcholine membranes. The phosphate group on the phospholipid plays a role in this interaction which may involve an electron transfer from the phosphate to the Fe^<3+>. A positively charged quaternary amine on the phosphatidylcholine is also necessary to give a peak absorbance at 610nm. Our observations are consistent with binding of one XO/Fe^<3+> complex to about 3 molecules of the egg yolk phosphatidylcholine carrying a zero net charge.