Hegstad A C, Strand H, Ytrehus K
Department of Medical Physiology, University of Tromsø, Norway.
J Mol Cell Cardiol. 1994 May;26(5):569-78. doi: 10.1006/jmcc.1994.1069.
Peroxidation of polyunsaturated fatty acids in cell membranes is thought to be a crucial factor in the cascade leading to reperfusion damage in the myocardium. However, some studies also describe increased lipid peroxidation in ischaemic tissue. The present study therefore examines phospholipid peroxidation after 60 min of global ischaemia and during the initial phase of reperfusion in isolated Langendorff-perfused rat hearts. Lipids were extracted from these hearts and separated into phospholipid, triglyceride and non-esterified fatty acid fractions. The phospholipid fraction was hydrolysed with phospholipase A2, and reverse-phase high performance liquid chromatography of the fatty acids derived from the phospholipids was performed. Peroxidized polyunsaturated fatty acids were separated from unchanged fatty acids and amounts of monohydroxy or monohydroperoxy isomers were quantified by measuring conjugated dienes by UV absorption (235 nm). Phospholipids from ischaemic as well as free-radical-exposed tissue contained increased levels of peroxidized polyunsaturated fatty acids (20.7 +/- 2.4 and 20.5 +/- 2.3 respectively, v 11.8 +/- 1.4 units/mg dry weight in controls). After 2-10 min of reperfusion, a significant increase in phospholipid peroxidation was no longer detected (12.5 +/- 1.2 units/mg). The amount and the composition of non-esterified fatty acids were examined by gas chromatography. Ischaemia significantly increased both the amount of non-esterified fatty acids (1.5 +/- 0.8 v 4.9 +/- 1.8 nmol/mg dry wt) as well as the percentage composed of arachidonic acid (3.4 +/- 3.2% v 7.4 +/- 1.4%). Fatty acid levels remained elevated during reperfusion (5.5 +/- 1.9 nmol/mg and 7.0 +/- 1.4%). In conclusion, our results have demonstrated that prolonged ischaemia alone caused phospholipid peroxidation as well as accumulation of non-esterified arachidonic acid. There was no sign of further phospholipid peroxidation during reperfusion.
细胞膜中多不饱和脂肪酸的过氧化被认为是导致心肌再灌注损伤级联反应中的一个关键因素。然而，一些研究也描述了缺血组织中脂质过氧化增加的情况。因此，本研究检测了离体Langendorff灌注大鼠心脏在全心缺血60分钟后及再灌注初始阶段的磷脂过氧化情况。从这些心脏中提取脂质，并分离为磷脂、甘油三酯和非酯化脂肪酸部分。用磷脂酶A2水解磷脂部分，并对源自磷脂的脂肪酸进行反相高效液相色谱分析。将过氧化的多不饱和脂肪酸与未改变的脂肪酸分离，并通过紫外吸收（235nm）测量共轭二烯来定量单羟基或单氢过氧基异构体的量。缺血组织以及暴露于自由基的组织中的磷脂含有增加水平的过氧化多不饱和脂肪酸（分别为20.7±2.4和20.5±2.3，对照组为11.8±1.4单位/毫克干重）。再灌注2 - 10分钟后，未检测到磷脂过氧化的显著增加（12.5±1.2单位/毫克）。通过气相色谱法检测非酯化脂肪酸的量和组成。缺血显著增加了非酯化脂肪酸的量（1.5±0.8对4.9±1.8纳摩尔/毫克干重）以及花生四烯酸所占的百分比（3.4±3.2%对7.4±1.4%）。再灌注期间脂肪酸水平仍然升高（5.5±1.9纳摩尔/毫克和7.0±1.4%）。总之，我们的结果表明，仅长时间缺血就会导致磷脂过氧化以及非酯化花生四烯酸的积累。再灌注期间没有进一步磷脂过氧化的迹象。
