Garcia Yngo J, Rodríguez-Malaver Antonio J, Peñaloza Nancy
Departamento de Bioquímica, Facultad de Medicina, Universidad de Los Andes, Mérida 5101, Venezuela.
J Neurosci Methods. 2005 May 15;144(1):127-35. doi: 10.1016/j.jneumeth.2004.10.018.
Lipid peroxidation by reactive oxygen species (ROS) is known to be involved in the damaging mechanism of several acute and chronic brain disorders. The most prominent and currently used assay as an index for lipid peroxidation products is the thiobarbituric acid assay (TBA test). It is based on the reactivity of an end product of lipid peroxidation, malondialdehyde (MDA) with TBA to produce a red adduct. However, it is known that the MDA levels are frequently overestimated, that the reaction lacks specificity and mainly reflects the susceptibility of brain tissue to the generation and degradation of newly formed lipid hydroperoxides under the TBA test conditions. The present paper shows that artifactual lipid peroxidation by TBA test conditions can be prevented and that the MDA level overestimation can be minimized in cerebellar slices. This can be done by incubating the slices in a continuous tissue perfusion system, by adding butylated hydroxytoluene (BHT) to the homogenization solutions and by carrying out the assay anaerobically on deproteinizated supernatants of cerebellar slice homogenates. The present research also showed that lipid peroxidation products generated during incubation of the slices by hydrogen peroxide (H2O2) could be measured without artifactual interference by the TBA test conditions.
已知活性氧（ROS）引发的脂质过氧化参与了多种急慢性脑部疾病的损伤机制。作为脂质过氧化产物指标，目前最常用且最突出的检测方法是硫代巴比妥酸检测法（TBA检测）。它基于脂质过氧化的终产物丙二醛（MDA）与TBA的反应生成红色加合物。然而，已知MDA水平经常被高估，该反应缺乏特异性，且在TBA检测条件下主要反映了脑组织对新形成的脂质氢过氧化物生成和降解的敏感性。本文表明，在小脑切片中，可以防止TBA检测条件下的人为脂质过氧化，并且可以将MDA水平的高估降至最低。这可以通过在连续组织灌注系统中孵育切片、在匀浆溶液中添加丁基羟基甲苯（BHT）以及对小脑切片匀浆的脱蛋白上清液进行厌氧检测来实现。本研究还表明，在切片经过氧化氢（H2O2）孵育期间产生的脂质过氧化产物可以在不受TBA检测条件人为干扰的情况下进行测量。
