Fung L W, Zhang Y
Department of Chemistry, Loyola University of Chicago, IL 60626.
Free Radic Biol Med. 1990;9(4):289-98. doi: 10.1016/0891-5849(90)90003-2.
The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When an oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in erythrocyte membranes, using fatty acid spin labels with nitroxide radicals on the hydrocarbon chains. About 50 microL of packed (about 5-6 x 10(8)), carbon monoxide (CO)-gassed red blood cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37 degrees C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3 +/- 1.8 x 10(-3)/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occurring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.
红细胞抵御细胞内氧化剂的防御系统复杂且高效。然而，细胞膜中产生的自由基相对地与细胞的抗氧化机制隔离开来。当氧化剂攻击超过红细胞抗氧化系统的能力时，可能会发生膜损伤，导致红细胞破坏和溶血性贫血。在本研究中，我们提出了一种使用烃链上带有氮氧自由基的脂肪酸自旋标记物来监测红细胞膜中自由基还原的方法。使用约50微升经一氧化碳（CO）充气的压积红细胞（约5 - 6×10⁸个）。在37℃下持续2小时，获取完整细胞中5 - 脱氧硬脂酸自旋标记物的电子顺磁共振信号随时间的变化情况。在我们的实验条件下，正常成年完整细胞中自旋标记物还原的伪一级速率常数为4.3±1.8×10⁻³/分钟。讨论了测量的重现性和变异性。由于我们所描述的测量反映了细胞膜中发生的自由基还原程度，我们认为该方法可用于测量抗氧化系统有缺陷的红细胞膜抵御氧化剂的能力。该方法对于测量药物、化学物质或环境毒素对抗氧化系统对膜中自由基的修饰作用特别有用。
