EVALUATION OF AN ISOLUMINOL CHEMI-LUMINESCENCE ASSAY FOR THE DETECTION OF HYDROPEROXIDES IN HUMAN-BLOOD PLASMA
EVALUATION OF AN ISOLUMINOL CHEMI-LUMINESCENCE ASSAY FOR THE DETECTION OF HYDROPEROXIDES IN HUMAN-BLOOD PLASMA
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DOI：
10.1016/0003-2697(88)90369-7
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发表时间：
1988-11-15
影响因子：
2.9
通讯作者：
AMES, BN
中科院分区：
文献类型：
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作者：
FREI, B;YAMAMOTO, Y;AMES, BN
Y.Y.Yamamoto，M.H.Brodsky，J.C.Baker和B.N.Ames(1987)最近报道了一种用高效液相色谱法和异鲁米诺化学发光法分离和检测生物样品中脂质过氧化氢和过氧化氢的方法(Y.Yamamoto，M.H.Brodsky，J.C.Baker和B.N.Ames(1987))。生物化学。160，7-13；Y.Y.Yamamoto和B.N Ames(1987)Free Rad.比奥尔。地中海医院。3,359-361)。本文详细介绍了该方法在人血浆分析中的应用，并对在用甲醇和正己烷提取的血浆中观察到的三种产生化学发光的化合物进行了进一步的表征。结果表明，该方法能检测到加入到血浆中的各种脂质过氧化氢。相反，加入到血浆中的过氧化氢会被内源性过氧化氢酶迅速降解。过氧化氢和第二种产生化学发光的次要化合物，分别出现在血浆的甲醇和己烷提取物的分析中，似乎是在分析过程中产生的，不太可能在血浆中存在。第三种化合物产生化学发光峰，被提取到血浆的正己烷相中，早期被归类为胆固醇酯氢过氧化氢，结果表明它既不是胆固醇酯，也不是氢过氧化氢，而是氢苯二酚泛喹酚-10。由于氢氧化钠或三苯基膦等还原试剂可消除过氧化氢的化学发光反应，而氢对苯二酚不能消除这种反应，因此应使用这种还原来确认检测中观察到的任何产生化学发光的脂质都是过氧化氢，而不是氢二酚。我们的结论是，从健康受试者分离的人血浆中，过氧化氢的浓度不太可能超过约0.25µm，也不含有浓度大于0.03µm的脂质过氧化氢。所述的方法在适当的预防措施下使用时，是一种方便和非常灵敏的生物组织中脂质过氧化氢的分析方法。
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 by 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 .mu.M and does not contain lipid hydroperoxides in concentrations greater than 0.03 .mu.M. The method described, when used with appropriate precautions, is a convenient and very sensitive assay for lipid hydroperoxides in biological tissues.