Frei B, Yamamoto Y, Niclas D, Ames B N
Department of Biochemistry, University of California, Berkeley 94720.
Anal Biochem. 1988 Nov 15;175(1):120-30. doi: 10.1016/0003-2697(88)90369-7.
An assay for the separation and detection of lipid hydroperoxides and hydrogen peroxide in biological samples using HPLC and isoluminol chemiluminescence was recently described (Y. Yamamoto, M. H. Brodsky, J. C. Baker, and B. N. Ames (1987) Anal. Biochem. 160, 7-13; Y. Yamamoto and B. N. Ames (1987) Free Rad. Biol. Med. 3, 359-361). In this paper the application of this assay to the analysis of human blood plasma is described in detail, and three compounds producing chemiluminescence that were observed in the initial studies in plasma extracted with methanol and hexane are further characterized. It is shown that various lipid hydroperoxides added to plasma are detected by the assay. In contrast, hydrogen peroxide added to plasma is rapidly degraded by endogenous catalase. Hydrogen peroxide and a second, minor compound producing chemiluminescence, which appear in the assay of the methanol and the hexane extract of plasma, respectively, appear to be generated during analysis and are not likely to be present in plasma. The third compound yielding a chemiluminescence peak, which is extracted into the hexane phase of plasma and was earlier assigned to cholesterol ester hydroperoxide, is shown to be neither a cholesterol ester nor a hydroperoxide, but the hydroquinone ubiquinol-10. As the chemiluminescence response of hydroperoxides, but not of hydroquinones, is eliminated by reducing reagents such as sodium borohydride or triphenylphosphine, such reduction should be used to confirm that any chemiluminescence producing lipid observed in the assay is a hydroperoxide, not a hydroquinone. We conclude that isolated human plasma from healthy subjects is very unlikely to contain hydrogen peroxide in concentrations greater than about 0.25 microM and does not contain lipid hydroperoxides in concentrations greater than 0.03 microM. The method described, when used with appropriate precautions, is a convenient and very sensitive assay for lipid hydroperoxides in biological tissues.
最近报道了一种使用高效液相色谱（HPLC）和异鲁米诺化学发光法分离和检测生物样品中脂质氢过氧化物和过氧化氢的分析方法（Y. 山本、M. H. 布罗德斯基、J. C. 贝克和B. N. 艾姆斯（1987年）《分析生物化学》160卷，第7 - 13页；Y. 山本和B. N. 艾姆斯（1987年）《自由基生物学与医学》3卷，第359 - 361页）。本文详细描述了该分析方法在人血浆分析中的应用，并对在用甲醇和己烷萃取的血浆初始研究中观察到的三种产生化学发光的化合物进行了进一步表征。结果表明，该分析方法能检测到添加到血浆中的各种脂质氢过氧化物。相比之下，添加到血浆中的过氧化氢会被内源性过氧化氢酶迅速降解。过氧化氢和另一种产生化学发光的次要化合物，分别出现在血浆甲醇提取物和己烷提取物的分析中，似乎是在分析过程中产生的，不太可能存在于血浆中。第三种产生化学发光峰的化合物，它被萃取到血浆的己烷相中，之前被认为是胆固醇酯氢过氧化物，结果表明它既不是胆固醇酯也不是氢过氧化物，而是对苯二酚泛醌 - 10。由于硼氢化钠或三苯基膦等还原剂可消除氢过氧化物而非对苯二酚的化学发光响应，因此应使用这种还原方法来确认分析中观察到的任何产生化学发光的脂质是氢过氧化物，而非对苯二酚。我们得出结论，健康受试者的分离人血浆中过氧化氢浓度极不可能高于约0.25微摩尔，脂质氢过氧化物浓度也不会高于0.03微摩尔。所述方法在采取适当预防措施时，是一种用于生物组织中脂质氢过氧化物的便捷且非常灵敏的分析方法。
