Genetics, Cell, and Developmental Biology Section, Department of Biology, University of Patras, Patras 26100, Greece.
Free Radic Biol Med. 2013 Jun;59:27-35. doi: 10.1016/j.freeradbiomed.2012.09.038. Epub 2012 Oct 3.
A simple and sensitive method is presented for the simultaneous quantification (spectrophotometric and spectrofluorimetric) of the main lipid and protein peroxidation products after their initial fractionation: free malondialdehyde (FrMDA), protein-bound malondialdehyde (PrMDA), total hydroperoxides (LOOH), and protein hydroperoxides (PrOOH). FrMDA and PrMDA (released from proteins by alkaline hydrolysis) are measured after the reaction of MDA with thiobarbituric acid (TBA) under acidic conditions, by the specific fluorimetric quantification of the resulting MDA-(TBA)2 adduct chromophore. The measurement of LOOH and PrOOH is based on the reaction of Fe(3+) (resulting from the reaction of LOOH and PrOOH with Fe(2+)) with xylenol orange (XO) and the photometric quantification of the resulting XO-Fe complex. The sensitivity of the assays for FrMDA/PrMDA and LOOH/PrOOH is 20 and 100pmol, respectively. The method was applied successfully on human plasma and can be used for the evaluation of oxidative stress in both basic and clinical research.
本文提出了一种简单而灵敏的方法，用于在初始分离后同时定量（分光光度法和荧光分光光度法）主要脂质和蛋白质过氧化产物：游离丙二醛（FrMDA）、蛋白质结合丙二醛（PrMDA）、总过氧化物（LOOH）和蛋白质过氧化物（PrOOH）。FrMDA 和 PrMDA（通过碱性水解从蛋白质中释放）在酸性条件下与硫代巴比妥酸（TBA）反应后，通过对生成的 MDA-（TBA）2 加合物发色团的特异性荧光定量来测量。LOOH 和 PrOOH 的测量基于 LOOH 和 PrOOH 与 Fe（2+）反应生成的 Fe（3+）（XO）与二甲苯酚橙（XO）的反应，以及生成的 XO-Fe 络合物的光密度定量。FrMDA/PrMDA 和 LOOH/PrOOH 测定的灵敏度分别为 20 和 100pmol。该方法已成功应用于人血浆，并可用于基础和临床研究中氧化应激的评估。
