Measurement of phosphatidylcholine hydroperoxides in solution and in intact membranes by the ferric-xylenol orange assay
Measurement of phosphatidylcholine hydroperoxides in solution and in intact membranes by the ferric-xylenol orange assay
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DOI：
10.1016/j.ab.2006.09.011
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发表时间：
2006-12-01
影响因子：
2.9
通讯作者：
Gebicki, Janusz M.
中科院分区：
文献类型：
--
作者：
Fukuzawa, Kenji;Fujisaki, Aya;Gebicki, Janusz M.
本文研究了三价铁与二甲酚橙子（XO）形成显色络合物测定过氧化氢（FOX法）。原始或修改的FOX方法是在由单一相组成的水溶液或有机溶液上进行的。然而，对于非均质样品（如生物材料）中的脂质过氧化物，大部分脂质被隔离在单独的相中。这些脂质的有机溶剂提取通常是不完全的，并且可能在提取过程中导致额外的过氧化。在这项研究中，我们应用FOX测定膜磷脂酰胆碱过氧化氢（PC-OOH）在分离相的测量。在60%MeOH中，蛋黄磷脂酰胆碱（EYPC）使Fe ~（3+）-XO络合物的560 nm宽峰移到610 nm，吸收强度增加，峰形变尖。峰的位移对于测量未知量的Fe 3+是有用的，因为未络合的XO对560 nm处的峰的吸光度有相当大的贡献，但不影响610 nm处的吸光度。需要EYPC形成膜以移动峰，因为在60% MeOH中发生了移动，但在用洗涤剂处理或在90% McOH中没有发生移动，其中EYPC没有形成膜。摩尔吸光系数（λ 610）为32，700 M-1 cm（-1），约为报道的摩尔吸光系数（λ 560）的两倍。我们将该方法应用于测定由EYPC制备的PC-OOH，并获得摩尔吸光系数（λ 610），在存在和不存在BHT的情况下分别为79，100和115，700 M-1 cm（-1）。这一发现允许测定PC-OOH浓度，即使在化学复杂的系统。(c)2006年爱思唯尔公司All rights reserved.
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 ran 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% McOH in which EYPC did not form the membranes. The molar absorption coefficient (epsilon 610) was 32,700 M-1 cm(-1), which was about twice the molar absorption co efficient (epsilon 560) reported. We applied this method to the assay of PC-OOH prepared from EYPC and obtained the molar absorption coefficients (epsilon 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. (c) 2006 Elsevier Inc. All rights reserved.