Measurement of lipid hydroperoxides by the ferric-xylenol orange method (1) characteristics of the ferric-xylenol orange/membrane phosphatidylcholine complex.
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 Fe2+ and formation of the colored ferric-xylenol orange (XO/Fe3+) product with a peak at 560 nm. The 560 nm absorbance peak of XO/Fe3+ shifts to a 610 nm 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/Fe3+-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 Fe3+. A positively charged quaternary amine on the phosphatidylcholine is also necessary to give a peak absorbance at 610 nm. Our observations are consistent with binding of one XO/Fe3+ complex to about 3 molecules of the egg yolk phosphatidylcholine carrying a zero net charge.
Faculty of Pharmacy, Yasuda Women's University, Hiroshima 731-0153, Japan. fukuzawa@yasuda-u.ac.jp
Kenji
K
Fukuzawa
Akira
A
Shibata
Chika
C
Okamura
Yuhki
Y
Fujiwara
Kaori
K
Akai
Koichiro
K
Tsuchiya
Akira
A
Tokumura
Janusz M
JM
Gebicki
D000588:Amines / Q000737:chemistry; D000818:Animals; D004530:Egg Yolk; D005227:Fatty Acids / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D005609:Free Radicals / Q000032:analysis*; D015227:Lipid Peroxidation; D008054:Lipid Peroxides / Q000032:analysis*; D008566:Membranes / Q000737:chemistry; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010636:Phenols; D010710:Phosphates / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000032:analysis*; D013454:Sulfoxides; D014992:Xylenes / Q000378:metabolism*
D000588:Amines; D005227:Fatty Acids; D005290:Ferric Compounds; D005456:Fluorescent Dyes; D005609:Free Radicals; D008054:Lipid Peroxides; D010636:Phenols; D010710:Phosphates; D010713:Phosphatidylcholines; D017382:Reactive Oxygen Species; D013454:Sulfoxides; D014992:Xylenes; C016833:xylenol orange