Kirkpatrick D T, Guth D J, Mavis R D
Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, NY 14642.
J Biochem Toxicol. 1986 Mar;1(1):93-104. doi: 10.1002/jbt.2570010110.
Thiobarbituric acid (TBA) assays which have been modified for detection of lipid hydroperoxides appear to be useful for demonstration of in vivo lipid peroxidation. Since these methods require heating tissue membranes with the buffered TBA, there is a possibility of interference from the detection of autoxidation that occurs during heating. These studies were undertaken to investigate conditions which favor TBA color production from hydroperoxide while limiting autoxidation during the assay. An acetic acid-sodium acetate buffered (pH 3.6) TBA assay was used. Heating linoleic acid hydroperoxide with 50 microM ferric iron or under nitrogen nearly doubled color production compared to heating it with no added iron or under air. The lipid antioxidant butylated hydroxytoluene inhibited color production from fatty acid hydroperoxides. When tissue fractions, including liver and lung microsomes and lung whole membranes, were heated in the assay, color production was greater under air than under nitrogen and was much greater under oxygen. When liver microsomes from carbon tetrachloride-exposed rats were used, color was increased only when oxygen was present in the heating atmosphere. The results with tissue fractions appear to demonstrate autoxidation during color development rather than the presence of preformed hydroperoxides. Finally, it was found that color production from membrane fractions was dependent on the vitamin E content of the membranes. It appears that autoxidation during heating should be limited by heating under nitrogen and not by adding antioxidants, which inhibit color production from hydroperoxides. As the vitamin E effect demonstrates, antioxidant status must be considered, since a change in color production could result from a change in antioxidant content without the accumulation of lipid hydroperoxides.
为检测脂质氢过氧化物而改良的硫代巴比妥酸（TBA）测定法，似乎可用于证明体内脂质过氧化。由于这些方法需要将组织膜与缓冲的TBA一起加热，因此存在加热过程中发生的自氧化检测干扰的可能性。进行这些研究是为了调查有利于从氢过氧化物产生TBA颜色，同时在测定过程中限制自氧化的条件。使用了乙酸 - 乙酸钠缓冲（pH 3.6）的TBA测定法。与不添加铁或在空气中加热相比，用50微摩尔铁或在氮气下加热亚油酸氢过氧化物，颜色产生几乎增加了一倍。脂质抗氧化剂丁基羟基甲苯抑制脂肪酸氢过氧化物的颜色产生。当包括肝和肺微粒体以及肺全膜在内的组织部分在测定中加热时，在空气中的颜色产生比在氮气下更大，在氧气下则大得多。当使用来自四氯化碳暴露大鼠的肝微粒体时，仅当加热气氛中有氧气时颜色才会增加。组织部分的结果似乎表明在显色过程中发生了自氧化，而不是存在预先形成的氢过氧化物。最后，发现膜部分的颜色产生取决于膜的维生素E含量。似乎加热过程中的自氧化应通过在氮气下加热来限制，而不是通过添加抑制氢过氧化物颜色产生的抗氧化剂。正如维生素E的作用所示，必须考虑抗氧化状态，因为颜色产生的变化可能是由于抗氧化剂含量的变化而导致的，而没有脂质氢过氧化物的积累。
