Cordis G A, Bagchi D, Maulik N, Das D K
University of Connecticut School of Medicine, Farmington 06030-1110.
J Chromatogr A. 1994 Feb 11;661(1-2):181-91. doi: 10.1016/0021-9673(94)85189-1.
Lipid peroxidation (LPO) is the oxidative deterioration of polyunsaturated fatty acids (PUFA) with the production of lipid hydroperoxides, cyclic peroxides, cyclic endoperoxides, and finally fragmentation to ketones and aldehydes (including malonaldehyde, MDA). Estimation of LPO through MDA formation measured by assaying thiobarbituric acid (TBA) reactive products remains the method of choice to study the development of oxidative stress in tissues. However, MDA estimation by TBA reactive products is non-specific and often gives erroneous results. In this report we describe a method using high-performance liquid chromatographic separation to estimate MDA, formaldehyde (FDA), acetaldehyde (ADA), acetone, and propionaldehyde (PDA), the degradation products of oxygen-derived free radicals (ODFR) and PUFA, as presumptive markers for LPO. Oxidative stress was induced in the tissue by perfusing an isolated rat heart with hydroxyl radical generating system (xanthine + xanthine oxidase + FeCl3 + EDTA). The coronary effluents were collected, derivatized with 2,4-dinitrophenylhydrazine (DNPH), and extracted with pentane. Aliquots of 25 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column. The products were eluted isocratically with a mobile phase containing acetonitrile-water-acetic acid (40:60:0.1, v/v/v), measured at three different wavelengths (307, 325 and 356 nm) using a Waters M-490 multichannel UV detector and collected for gas chromatography-mass spectrometry (GC-MS) analysis. The peaks were identified by cochromatography with DNPH derivatives of authentic standards, peak addition, UV pattern of absorption at the three wavelengths, and by GC-MS. The retention items of MDA, FDA, ADA, acetone, and PDA were 5.3, 6.6, 10.3, 16.5, and 20.5 min, respectively. The results of our study indicated progressive increase of all five lipid metabolites as a function of the duration of ODFR perfusion. Hydroxyl radical scavengers, superoxide dismutase plus catalase, completely inhibited the formation of these lipid metabolites, demonstrating that the release of lipid metabolites from the isolated heart was indeed in response to oxidative stress. Since MDA, FDA, ADA, acetone, and PDA are the products of ODFR-PUFA interactions, this method allows proper estimation of LPO which monitors the oxidative stress developed during the reperfusion of ischemic myocardium.
脂质过氧化（LPO）是多不饱和脂肪酸（PUFA）的氧化降解过程，会产生脂质氢过氧化物、环状过氧化物、环状内过氧化物，最终分解为酮和醛（包括丙二醛，MDA）。通过检测硫代巴比妥酸（TBA）反应产物来测定MDA从而估算LPO，仍然是研究组织中氧化应激发展的首选方法。然而，通过TBA反应产物估算MDA是非特异性的，并且常常得出错误结果。在本报告中，我们描述了一种使用高效液相色谱分离法来估算MDA、甲醛（FDA）、乙醛（ADA）、丙酮和丙醛（PDA）的方法，这些物质是氧衍生自由基（ODFR）和PUFA的降解产物，作为LPO的推定标志物。通过用羟自由基生成系统（黄嘌呤+黄嘌呤氧化酶+FeCl3+EDTA）灌注离体大鼠心脏，在组织中诱导氧化应激。收集冠状动脉流出液，用2,4-二硝基苯肼（DNPH）衍生化，并用戊烷萃取。取25微升乙腈溶液注入到贝克曼Ultrasphere C18（3微米）柱上。产物用含有乙腈-水-乙酸（40:60:0.1，v/v/v）的流动相等度洗脱，使用沃特斯M-490多通道紫外检测器在三个不同波长（307、325和356纳米）处进行测定，并收集用于气相色谱-质谱（GC-MS）分析。通过与标准品的DNPH衍生物共色谱、峰添加、三个波长处的紫外吸收图谱以及GC-MS来鉴定峰。MDA、FDA、ADA、丙酮和PDA的保留时间分别为5.3、6.6、10.3、16.5和20.5分钟。我们的研究结果表明，随着ODFR灌注时间的延长，所有五种脂质代谢产物都逐渐增加。羟自由基清除剂超氧化物歧化酶加过氧化氢酶完全抑制了这些脂质代谢产物的形成，表明离体心脏中脂质代谢产物的释放确实是对氧化应激的反应。由于MDA、FDA、ADA、丙酮和PDA是ODFR-PUFA相互作用的产物，这种方法能够对LPO进行适当估算，从而监测缺血心肌再灌注过程中产生的氧化应激。
