Benedetti A, Esterbauer H, Ferrali M, Fulceri R, Comporti M
Biochim Biophys Acta. 1982 May 13;711(2):345-56. doi: 10.1016/0005-2760(82)90044-3.
Since it has been demonstrated in previous studies that peroxidation of liver microsomal lipids leads to the production of aldehydes provided with cytopathological activities--namely 4-hydroxyalkenals--evidence was searched for aldehydes bound to microsomal protein in in vivo conditions (CCl4 and BrCCl3 intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been demonstrated previously. The spectrophotometric analysis of 2,4-dinitrophenylhydrazine-treated non-lipoidal residues of liver microsomes from the intoxicated rats shows absorption spectra similar to those observed for the dinitrophenylhydrazones formed in the reaction of alkenals with -SH groups of proteins or low molecular weight thiols. Similar spectra, although magnified from a quantitative point of view, were obtained either with liver microsomes allowed to react with synthetic 4-hydroxynonenal or with liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation shows that the amount of 2,4-dinitrophenylhydrazine-reacting groups in the non-lipoidal residue of liver microsomes increases with the incubation time and is correlated to the amount of thiobarbituric acid-reacting products formed in the incubation mixture. In both the in vivo conditions (CCl4 and BrCCl3 intoxications) the amount of 2,4-dinitrophenylhydrazine-reacting groups in the non-lipoidal residue of liver microsomes increases from 15 min up to 2 h after poisoning and is higher, in every instance, in the BrCCl3-intoxicated animals compared to the CCl4-poisoned ones. Experiments carried out to ascertain the reliability of the spectrophotometric detection of protein-bound alkenals showed that in the in vitro system in which liver microsomes are allowed to react with 4-hydroxynonenal there is a good agreement between the binding value that can be calculated from the absorption spectrum and the binding value obtained by using labelled 4-hydroxynonenal.
由于先前的研究已证明肝微粒体脂质的过氧化会导致具有细胞病理活性的醛类物质（即4-羟基烯醛）的产生，因此在体内条件下（四氯化碳和三氯溴甲烷中毒）寻找与微粒体蛋白结合的醛类物质的证据，此前已证明肝内质网脂质发生了过氧化。对中毒大鼠肝脏微粒体经2,4-二硝基苯肼处理的非脂质残留物进行分光光度分析，结果显示其吸收光谱与烯醛与蛋白质或低分子量硫醇的-SH基团反应形成的二硝基苯腙的吸收光谱相似。无论是让肝脏微粒体与合成的4-羟基壬烯醛反应，还是在NADPH-铁依赖系统中使肝脏微粒体发生过氧化，都能得到类似的光谱，尽管从定量角度看有所放大。对微粒体脂质过氧化的时间进程研究表明，肝脏微粒体非脂质残留物中与2,4-二硝基苯肼反应的基团数量随孵育时间增加，且与孵育混合物中形成的硫代巴比妥酸反应产物的量相关。在两种体内条件下（四氯化碳和三氯溴甲烷中毒），肝脏微粒体非脂质残留物中与2,4-二硝基苯肼反应的基团数量在中毒后15分钟至2小时内增加，并且在每种情况下，三氯溴甲烷中毒动物中的该数量均高于四氯化碳中毒动物。为确定分光光度法检测与蛋白结合的烯醛的可靠性而进行的实验表明，在肝脏微粒体与4-羟基壬烯醛反应的体外系统中，根据吸收光谱计算出的结合值与使用标记的4-羟基壬烯醛获得的结合值之间具有良好的一致性。
