Thomas J P, Kalyanaraman B, Girotti A W
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Arch Biochem Biophys. 1994 Dec;315(2):244-54. doi: 10.1006/abbi.1994.1496.
Oxidative modification of human low-density lipoprotein (LDL) is thought to play an important role in the development of atherosclerosis. LDL oxidizability is believed to be strongly influenced by factors such as (a) content of preexisting lipid hydroperoxides (LOOHs) and (b) content of endogenous antioxidants such as alpha-tocopherol and beta-carotene. The purpose of this study was to examine the prooxidant role of preexisting LDL-LOOHs, using a recently developed method for ultrasensitive and selective LOOH analysis: high-performance liquid chromatography with mercury drop electrochemical detection (HPLC-EC). Exceedingly low detection limits for LDL-LOOHs have been achieved by HPLC-EC, e.g., approximately 100 fmol for cholesteryl ester hydroperoxide (CEOOH). This sensitivity has allowed us to monitor LDL-LOOHs at levels that are undetectable by most other methods. Fresh LDL prepared with the utmost care to prevent autoxidation was found to contain small, yet significant amounts of CEOOH, 6-12 pmol/mg protein. Our data suggest that these peroxides could not have arisen during LDL isolation or sample work-up for HPLC-EC. Incubation with GSH and phospholipid hydroperoxide glutathione peroxidase resulted in nearly complete reduction of the CEOOH. This LDL was found to be much more resistant to Cu(2+)-induced peroxidation than starting material, exhibiting a lag period that was at least six times greater. We have also determined that LDL becomes progressively more susceptible to Cu(2+)-induced lipid peroxidation (as evidenced by a shortened lag) when it is preloaded with increasing amounts of photochemically generated LOOHs. Taken together, these results provide strong support for the idea that preexisting LOOHs in LDL are important determinants of its overall oxidizability.
人类低密度脂蛋白（LDL）的氧化修饰被认为在动脉粥样硬化的发展中起重要作用。LDL的氧化能力被认为受到多种因素的强烈影响，例如：（a）预先存在的脂质氢过氧化物（LOOHs）的含量，以及（b）内源性抗氧化剂如α-生育酚和β-胡萝卜素的含量。本研究的目的是使用最近开发的超灵敏和选择性LOOH分析方法：汞滴电化学检测高效液相色谱法（HPLC-EC），来研究预先存在的LDL-LOOHs的促氧化作用。HPLC-EC已实现对LDL-LOOHs极低的检测限，例如，胆固醇酯氢过氧化物（CEOOH）约为100飞摩尔。这种灵敏度使我们能够监测到大多数其他方法无法检测到的LDL-LOOHs水平。在极其小心地制备以防止自氧化的新鲜LDL中，发现含有少量但显著量的CEOOH，为6 - 12皮摩尔/毫克蛋白质。我们的数据表明，这些过氧化物不可能在LDL分离或用于HPLC-EC的样品处理过程中产生。与谷胱甘肽（GSH）和磷脂氢过氧化物谷胱甘肽过氧化物酶一起孵育导致CEOOH几乎完全还原。发现这种LDL比起始材料对铜离子（Cu(2+)）诱导的过氧化更具抗性，表现出至少大六倍的延迟期。我们还确定，当LDL预先加载越来越多光化学产生的LOOHs时，它对Cu(2+)诱导的脂质过氧化变得越来越敏感（表现为延迟期缩短）。综上所述，这些结果为LDL中预先存在的LOOHs是其整体氧化能力的重要决定因素这一观点提供了有力支持。
