Tuma D J, Thiele G M, Xu D, Klassen L W, Sorrell M F
VA Alcohol Research Center, Department of Veterans Affairs Medical Center, Omaha, NE, USA.
Hepatology. 1996 Apr;23(4):872-80. doi: 10.1002/hep.510230431.
Acetaldehyde and the lipid peroxidation-derived aldehyde malondialdehyde (MDA), are reactive compounds that are generated during ethanol metabolism in the liver, and both aldehydes have been shown to be capable of binding to proteins and forming stable adducts. Because similar concentrations of MDA and acetaldehyde can coexist in the liver during ethanol oxidation, protein adduct formation in the presence of both of these aldehydes was studied under both in vitro and in vivo conditions. When proteins were incubated in the presence of both MDA and acetaldehyde, MDA caused a marked and concentration-dependent increase in the stable binding of acetaldehyde to proteins. Maximum stimulation of binding occurred at approximately a fourfold molar excess of MDA relative to acetaldehyde when concentrations of 1.0 mmol/L and 0.1 mmol/L were tested. The formation of highly fluorescent product or products was associated with the MDA stimulation of acetaldehyde binding, indicating that new and distinct products were being generated. These hybrid adducts of MDA and acetaldehyde have been designated as MAA adducts. An affinity-purified polyclonal antibody was produced that specifically recognized MAA epitopes on proteins and did not cross-react with carrier proteins or proteins modified with either acetaldehyde or MDA alone. A quantitative competitive enzyme-linked immunosorbent assay (ELISA) was developed and detected the presence of MAA-modified proteins in liver cytosol from ethanol-fed rats but not in pair-fed controls. Quantification of the data from the competitive ELISA indicated the presence of approximately 75 pmoles protein-bound MAA per milligram liver cytosol proteins of the ethanol-fed animals. These results indicate that acetaldehyde and MDA can react together in a synergistic manner and generate hybrid adducts (MAA-adducts) and further suggest that MAA adducts may represent a major species of adducts formed in the liver during ethanol metabolism in vivo.
乙醛和脂质过氧化衍生的醛丙二醛（MDA）是肝脏乙醇代谢过程中产生的反应性化合物，这两种醛均已被证明能够与蛋白质结合并形成稳定的加合物。由于在乙醇氧化过程中，肝脏中MDA和乙醛能以相似的浓度共存，因此我们在体外和体内条件下研究了这两种醛同时存在时蛋白质加合物的形成情况。当蛋白质在MDA和乙醛同时存在的情况下孵育时，MDA会导致乙醛与蛋白质的稳定结合显著增加，且呈浓度依赖性。当测试浓度为1.0 mmol/L和0.1 mmol/L时，相对于乙醛，MDA的摩尔过量约为四倍时，结合的刺激作用达到最大。高荧光产物的形成与MDA对乙醛结合的刺激作用相关，表明正在生成新的、独特的产物。这些MDA和乙醛的混合加合物被命名为MAA加合物。制备了一种亲和纯化的多克隆抗体，该抗体能特异性识别蛋白质上的MAA表位，且不与载体蛋白或仅用乙醛或MDA修饰的蛋白质发生交叉反应。开发了一种定量竞争性酶联免疫吸附测定（ELISA）方法，该方法检测到乙醇喂养大鼠肝脏细胞溶质中存在MAA修饰的蛋白质，而在配对喂养的对照中未检测到。对竞争性ELISA数据的定量分析表明，乙醇喂养动物每毫克肝脏细胞溶质蛋白中约存在75皮摩尔与蛋白质结合的MAA。这些结果表明，乙醛和MDA可以协同反应并生成混合加合物（MAA加合物），进一步表明MAA加合物可能代表体内乙醇代谢过程中肝脏中形成的主要加合物种类。
