Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.

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SummaryThe 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 ϩ and formation of the colored ferric-xylenol orange (XO/Fe 3 ϩ ) product with a peak at 560 nm. The 560 nm absorbance peak of XO/Fe 3 ϩ 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/Fe 3 ϩ -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 ϩ . 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/Fe 3 ϩ complex to about 3 molecules of the egg yolk phosphatidylcholine carrying a zero net charge.

SummaryThe ferric-xylenol orange (FOX) method has been used for quantification of hydroperoxides by measuring the colored ferric-xylenol orange (XO/Fe 3 ϩ ) product with a peak absorbance at 560 nm. We recently reported a modified FOX method, the sensitivity of which was increased in the presence of membranous phosphatidylcholine (PC) by forming a XO/Fe 3 ϩ -PC complex with a peak absorbance at 610 nm. Lipoxygenases (LOXs) and their metabolites have been implicated in a wide range of disease states. We applied our newly developed FOX method to the assay of human 15-lipoxygenase-2 (15-LOX-2) and soybean lipoxygenase (SLOX) as typical animal and plant lipoxygenases, respectively. The amounts of 15-S -hydroperoxyeicosa-5,8,10,14-tetraenoic acid (15-HPETE) produced by 15-LOX-2 measured by UV-absorption at 237 nm attributed to the conjugated diene, coincided with the results of our FOX method measuring absorbance at 610 nm. The 15-HPETE production time courses measured by the two methods also correlated well. SLOX rapidly oxidized unesterified linoleic acids (LA) and slowly esterified fatty acids in egg yolk PC (EYPC). Availability of EYPC was increased if the membrane structure was moderately disturbed by MeOH and Triton X-100, but LA oxidation was readily decreased by them. These results indicate that our method is useful for lipoxygenase assay. Furthermore, our method was applicable to assaying the inhibitory effect of 5,8,11,14-eicosatetraynoic acid (ETYA) on SLOX activity using LA and EYPC as substrates. The inhibition dose-dependency of ETYA was almost the same in the LA and EYPC systems, although the enzyme concentrations differed by a factor of 1,000, suggesting that ETYA functioned as a competitive inhibitor. These results indicate that our method may be useful as a screen for the identification of novel inhibitors of lipoxygenases.

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