NADPH-dependent lipid peroxidation capacity in unfixed tissue sections: characterization of the pro-oxidizing conditions and optimization of the histochemical detection.
M Thomas, W M Frederiks, C J Van Noorden, K S Bosch, A Pompella
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引用次数: 0
Abstract
Factors which influence the iron-stimulated lipid peroxidation in rat liver have been studied by incubating unfixed cryostat sections with a pro-oxidant system and using an optimized histochemical detection method for lipid peroxidation products with 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B. We used a method that was slightly different from the one described previously. The final reaction product was exclusively localized in the cytoplasm of liver parenchymal cells with a homogeneous distribution within the liver lobule. The absorbance maximum, as measured cytophotometrically, was found to be 550 nm. Maximum lipid peroxidation was observed when the pro-oxidant system contained 0.2 mM NADPH, 1 mM ADP and 15 microM FeCl2. Some reaction product was found when NADPH was omitted. Iron concentrations higher than 180 microM prevented the formation of lipid peroxidation products in certain areas of the sections, whereas ADP concentrations higher than 1 mM inhibited the reaction in the whole section. A pH dependency was also observed, with the highest lipid peroxidation at pH 7.2. Optimum lipid peroxidation was induced by incubating for 30 min at 37 degrees C with the pro-oxidant system. A linear relationship was found between the thickness of the sections (up to 20 microns) and the amount of lipid peroxidation products. The addition of scavengers of O2-. (superoxide dismutase), hydrogen peroxide (catalase) and OH. (mannitol) to the first step medium did not affect the amount of final reaction product. These findings appear to confirm the hypothesis proposed for events occurring in isolated microsomes, leading to the formation of hydroperoxides and ultimately lipid peroxidation-derived carbonyls.(ABSTRACT TRUNCATED AT 250 WORDS)
影响铁刺激的大鼠肝脏脂质过氧化的因素通过在促氧化系统中培养未固定的低温恒温器切片,并使用优化的脂质过氧化产物的组织化学检测方法(3-羟基-2-萘酸肼和Fast Blue b)进行了研究。我们使用的方法与之前描述的方法略有不同。最终反应产物完全定位于肝实质细胞的细胞质中,在肝小叶内均匀分布。用细胞光度法测得吸光度最大值为550 nm。当促氧化体系中含有0.2 mM NADPH、1 mM ADP和15微米FeCl2时,脂质过氧化作用最大。当忽略NADPH时,发现了一些反应产物。铁浓度高于180 μ m可抑制部分区域脂质过氧化产物的形成,而ADP浓度高于1 μ m可抑制整个区域的反应。还观察到pH依赖性,pH为7.2时脂质过氧化率最高。促氧化体系在37℃下培养30 min,可诱导脂质过氧化。切片的厚度(可达20微米)与脂质过氧化产物的量之间存在线性关系。O2-清除剂的加入。(超氧化物歧化酶),过氧化氢(过氧化氢酶)和OH。(甘露醇)加到第一步培养基中不影响最终反应产物的量。这些发现似乎证实了在分离微粒体中发生的事件的假设,这些事件导致氢过氧化物和最终脂质过氧化衍生羰基的形成。(摘要删节250字)