直接法测定低密度脂蛋白胆固醇

为了提高血脂检验技术,探讨直接法测定低密度脂蛋白胆固醇(LDL-C),选用市售的两种直接测定LDL-C的试剂盒进行方法学的实验评价,结果表明：直接法具有简便、快速、准确和适合自动分析等特点.两种试剂盒的测定结果都能满足建立分析方法要求的目标,不精密度＜４％,两法显著相关r=0.994, y=1.0572x－0.1052, S_y/x=0.1913,且各种方法学检验结果相近,使用者可结合实际选作常规测定之用.     

极低密度脂蛋白受体研究进展

极低密度脂蛋白受体 (VLDL R)属于低密度脂蛋白受体 (LDL R)超家族 ,结构上与LDL R极为相似 ,而在结合特性、组织分布及生理功能上存在较大的差异 .近年来的研究表明 ,VLDL R配体结合域中的重复序列参与配体的结合 ,并已初步确定受体N端的 4个重复序列中含有与配体结合的重要位点 ;在哺乳动物体内 ,VLDL R主要分布于脂代谢活跃的组织细胞 ,如 :心肌、骨骼肌和脂肪组织等 ,表明其与脂质尤其是甘油三酯的代谢密切相关 ;动脉粥样硬化 (AS)的病变斑块组织中该受体的表达量很高 ,推测VLDL R参与了AS的病变过程 ;在不同的细胞内 ,VLDL R及其亚型的表达并不一致 ,并发现与各种生理、病理变化相关 ,已经发现多种转录因子参与VLDL R表达的调控 ;基因敲除研究也不断揭示VLDL R新的功能意义 ,特别是发现了VLDL R在脑的发育过程中的重要信号作用 ,这些研究已使人们对VLDL R有了新的更全面的认识     

Glucose-Modified Low Density Lipoprotein Enhances Human Monocyte Chemotaxis

In diabetes mellitus the progression of atherosclerosis is accelerated. The interaction of glucose with athero-genic lipoproteins may be relevant to the mechanisms responsible for this vascular damage. The aim of this study was to examine the effect of glucose-modified low density lipoprotein (LDL) on human monocyte chemotaxis and to investigate the roles of oxidation and glycation in the generation of chemotactic LDL. Cu(II)-mediated LDL oxidation was potentiated by glucose in a dose-dependent manner and increased its chemotactic activity. Incubation with glucose alone, under conditions where very little oxidation was observed, also increased the chemotactic property of LDL. Neither diethylenetriamine pentaacetic acid (DETAPAC) nor aminoguanidine, which both inhibited LDL oxidation, completely inhibited the chemotactic activity of glycated oxidised LDL. The results suggest that both oxidation and glycation contribute to increased chemotactic activity.     

Oxidized Low Density Lipoprotein and Inflammation in Gout Patients

To analyze the levels of oxidized low density lipoprotein (ox-LDL) and inflammatory cytokines in the plasma of gout patients. The levels of ox-LDL, hypersensitive C-reactive protein (hs-CRP), interleukin-1β, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured in the plasma of 41 gout patients [28 in acute phase episode, 13 in intermittent phase (IP)], and in 40 healthy controls. The relationship between ox-LDL and inflammation was also explored by measuring the levels of several pro-inflammatory cytokines in the plasma. The plasma levels of ox-LDL, hs-CRP, IL-6 and TNF-α were significantly increased in patients with gout in the acute phase compared to those in the IP group and healthy controls (P < 0.05), but the levels of TGF-β were significantly lower in the acute phase group than in the IP group and healthy controls (P < 0.01). The levels of ox-LDL in the gout patients in the IP were significantly higher than those in healthy controls (P < 0.05). Correlation analysis indicated that the levels of ox-LDL were positively correlated with hs-CRP, IL-6 and TNF-α (r = 0.343, r = 0.386, r = 0.659, P < 0.01, respectively), but negatively correlated with TGF-β levels in patients in the acute phase (r = ?0.240, P < 0.05). The levels of ox-LDL in gout patients were significantly higher than those in healthy controls. The changes in ox-LDL levels may be associated with enhanced inflammation in gout patients.     

LOX-1与动脉粥样硬化

氧化低密度脂蛋白(oxidizedlow-densitylipoprotein,Ox-LDL)是修饰脂蛋白中的一种,其与动脉粥样硬化的发生发展密切相关。1997年,Sawamura等在牛主动脉内皮细胞上发现了Ox-LDL的一种新型受体,即植物凝集素样氧化型低密度脂蛋白受体-1(lectin-likeoxidizedlowdensitylipoproteinreceptor-1,LOX-1),其在结构和功能上不同于其他类型的巨噬细胞清道夫受体。该文介绍LOX-1在近几年来的一些研究新进展,及其与动脉粥样硬化发病机制间的相互关系。     

Investigation of Lipid Peroxidation in Human Low Density Lipoprotein

Human plasma low density lipoprotein (LDL) exposed to oxygen saturated buffer becomes depleted of alpha-tocopherol within 3 to 6 hours. Thereafter, lipid peroxidation commences as evidenced by the loss of 18:2 (67nmol/mg LDL) and 20:4 (12nmol/mg LDL) and the concomitant formation of 4-hydroxy-nonenal (0.28 nmol/mg LDL) and fluorescent compounds. The major fluorophor in apo B of oxidized LDL has an excitation maximum at 355 nm and an emission maximum at 430 nm. A fluorophor with the same spectral properties is produced in apo B, if LDL is incubated with 4-hydroxynonenal, whereas malonal-dehyde gives a fluorophor with excitation and emission maxima at 400/470nm. Three-dimensional fluorescence spcetroscopy proved to be an useful tool in analysing the complex fluorescence of apo B.     

Seasonal Variation in Low Density Lipoprotein Oxidation and Antioxidant Status

Accumulating evidence indicates that oxidative modification of low-density lipoproteins is atherogenic and that antioxidants may play a role in protection of LDL against oxidation. Several studies have reported a seasonal fluctuation in antioxidant levels, but to date nothing is known about seasonal fluctuations in parameters of oxidizability. We collected blood from 10 volunteers at four different periods over one year (February, May, September and December), and measured the amount of plasma lip ids, plasma antioxidants, lipid and fatty acid composition of the LDL particle, LDL antioxidant content, LDL particle size and oxidation parameters (lag time and propagation rate). No seasonal fluctuation for lag time and propagation rate of copper ion-induced LDL oxidation was found. Small seasonal fluctuations were observed for some determinants of LDL oxidation, e.g. plasma and LDL vitamin E and LDL particle size, and for plasma lipids, plasma and LDL lutein and LDL p-carotene. Fatty acid composition of LDL did not change during the year. The main determinant of oxidation susceptibility was the fatty acid composition of LDL. We conclude that LDL oxidation parameters do not change over the year.     

巨噬细胞新型氧化低密度脂蛋白结合蛋白的研究

小鼠腹腔巨噬细胞（ＭＰＭ）膜上存在能结合氧化低密度脂蛋白（ｏｘ－ＬＤＬ）的Ａ类清道夫受体（ＳＲ－Ａ）,但用配体印迹技术研究制备ＭＰＭ膜蛋白,发现还存在一种新型ｏｘ－ＬＤＬ膜结合蛋白,其分子量低于ＳＲ－Ａ,为９２ｋＤ它不结合乙酰化低密度脂蛋白ａｃ－ＬＤＬ）,与配体的结合也不受还原剂的影响,但唾液酸酶处理则明显减弱其与ｏｘ－ＬＤＬ的结合,未标记ｏｘ－ＬＤＬ能竞争性抑制（＾１２５Ｉ）ｏｘ－ＬＤＬ与９２ｋ     

氧化修饰极低密度脂蛋白增强其致动脉粥样硬化作用

研究了氧化修饰极低密度脂蛋白（ｏｘ－ＶＬＤＬ）对小白鼠腹腔巨噬细胞内脂质堆积作用及其机制。经Ｃｕ~（２＋）修饰后ＶＬＤＬ的电泳迁移率及脂质过氧化物含量均显著增加。ｏｘ－ＶＬＤＬ更易导致小鼠腹腔巨噬细胞内脂质堆积。以相同浓度（３００μｇＴＧ／ｍＬ）或不同浓度（２００─５００μｇＴＧ／ｍＬ）的ｏｘ－ＶＬＤＬ及正常ＶＬＤＬ（ｎ－ＶＬＤＬ）与巨噬细胞温育２４ｈ,前者使巨噬细胞内ＴＧ堆积均比后者显著（Ｐ＜０．０１）。同时,随ｏｘ－ＶＬＤＬ的脂质过氧化物含量（ＴＢＡＲＳ水平）增加,巨噬细胞内ＴＧ含量的百分率相应增加。以５０μｇ蛋白／ｍＬ的ｎ－ＬＤＬ,ｏｘ－ＬＤＬ,ｎ－ＶＬＤＬ及ｏｘ－ＶＬＤＬ与巨噬细胞温育６０ｈ。细胞内ＣＥ堆积中氧化组均比正常组高（Ｐ＜０．０１）。巨噬细胞对~（１２５）Ｉ－ｎ－ＶＬＤＬ与~（１２５）Ｉ－ｏｘ－ＶＬＤＬ的结合、降曲线均有饱和趋势。两结合曲线无明显差异,但细胞对后者降解的量比前者多。结合的竞争实验表明,ｎ－ＶＬＤＬ能抑制大部分~（１２５）Ｉ－ｏｘ－ＶＬＤＬ与细胞结合,而Ａｃ－ＬＤＬ只能抑制小部分。结果表明ｏｘ－ＶＬＤＬ主要通过受体途径：大部分经过ｎ－ＶＬＤＬ受体,小部分经过清道夫受体被巨噬细胞摄     

维生素C抑制低密度脂蛋白的氧化修饰

研究了不同浓度维生素 C 对 Cu²⁺诱导的低密度脂蛋白(LDL)氧化修饰的抑制作用,通过测定硫代巴比妥酸反应物质(TBARS),荧光物质(lipofusion)扫描及琼脂糖电泳,显示一定浓度的维生素 C 在24h 内对 LDL 的氧化修饰具有抑制作用,并呈现量效效应.提示维生素 C 作为体内存在的一种抗氧化物,可抑制 LDL的氧化修饰,从而在防治动脉粥样硬化的发生具有一定意义.     

Antioxidant Activity of Ubiquinone-3 in Human Low Density Lipoprotein

The ability of ubiquinone-3, a short chain ubiquinone homologue, to prevent Cu²⁺ induced oxidation of human low density lipoprotein was investigated. The results are as follows: in the presence of ubiquinone-3 the extent of peroxidation, as determined by the formation of thiobarbituric acid reactive substances, was only one third of that found in its absence; the quinone can also prevent the fragmentation of apolipo-protein B-100 and the increase of the net negative surface charge of the particle.     

Evidence for Protein Subunits in Low Density Lipoprotein from Pig Serum

The two low density lipoproteins (LDL₁ and LDL₂) in pig serum were isolated and their aqueous solutions extracted with ethyl ether. On ultracentrifugation four components of 13, 20, 24 and 30 S were evident in both fractions, and occasionally a fifth of 10 S could be detected. The 13 S component represented about 70% of the extracted material and its protein moiety was estimated to be half that of the parent lipoprotein. This shows that both LDL fractions contain protein subunits that dissociate on lipid extraction, and these apparently reassociate to form the heavier components. Apart from lipid content the only difference between LDL₁ and LDL₂ was a small, but statistically significant, difference in the histidine content.     

Oxidation of Low Density Lipoprotein Upon Sequential Exposure to Copper Ions

Copper-induced LDL oxidation is characterized by an 'induction phase' (lag phase) during which the endogenous antioxidants are consumed, followed by a 'propagation phase' in which the LDL-associated polyunsaturated fatty acids are oxidized. Oxidation products may play an important role in the propagation of the oxidative process in the arterial intima as they increase the permeability of the damaged endothelium to various plasma components, including LDL. We therefore found it of interest to investigate the kinetics of LDL oxidation in vitro under conditions where LDL is sequentially exposed to Cu²⁺-induced oxidation.

The results of our studies demonstrate that when native LDL is exposed to copper oxidation in a medium containing oxidized LDL, oxidation of the added LDL may be almost instantaneous. Furthermore, even when native LDL is added to 'oxidizing LDL' towards the end of the lag phase or during the propagation phase it becomes oxidized after a very short lag. This oxidation process, occurring in spite of the possible protective effect of the antioxidants present in the newly added LDL, indicates that although antioxidants prolong the latency period by preventing the formation of active free radicals, when such radicals are present in the system, oxidation propagates. These results lend strong support to the generally accepted paradigm regarding the mechanism of propagation of lipid oxidation.

In view of the effect of oxidation products on the permeability of the endothelium, the observed shortening of the lag period may result in a vicious cycle, independent of the LDL-associated antioxidants, leading to continuing oxidation and foam cell formation.     

巨噬细胞极低密度脂蛋白受体的调节作用

本文研究了小鼠腹腔巨噬细胞极低密度脂蛋白(VLDL)受体的调节。用富含甘油三酯(TG)的VLDL与小鼠巨噬细胞预温育后,细胞结合~(125)I-VLDL的最大结合容量(Bmax)比对照细胞只降低5％(1071/1127ng/mg细胞蛋白质),当细胞内TG增加到对照的2.5倍时,细胞摄取及降解~(125)I-VLDL的量分别下降40％和22％;乙酰-低密度脂蛋白(AC-LDL)预温育的细胞结合~(125)I-VLDL的Bmax比对照细胞只降低14％(633/831ng/mg细胞蛋白质),当细胞内胆固醇(Ch)增加到对照的23倍时,细胞摄取及降解~(125)I-VLDL的量只分别下降10％和21％。此后随着细胞内TG或Ch含量的增加,摄取及降解~(125)I-VLDL的量仍保持不变。 结论:细胞内TG或Ch含量对VLDL受体的调节作用微弱,与TG相比,Ch的调节作用更弱。     

Diphenylhexatriene as a Fluorescent Probe for Monitoring Low Density Lipoprotein Peroxidation

The use of the fluorescent probe diphenylhexatriene (DPH) for monitoring low density lipoprotein (LDL) peroxidation has been investigated. The DPH incorporation into LDL results in a high fluorescence signal which decreases with time after addition of cupric ions. A strong correlation was found between the decay of the DPH fluorescence signal and the appearance of the thiobarbituric reactive substances (TBARS). HPLC and spectrofluorometric analyses demonstrated that DPH is destroyed during the time course of the copper-induced LDL peroxidation. The decrease in DPH fluorescent signal is prevented by addition of EDTA, vitamin E and drugs which protect LDL against peroxidation such as probucol or calcium antagonists. The high fluorescence of DPH allows the use of very small quantities of LDL (less than 5 μg/ml LDL protein). We thus suggest that DPH could be of use for continuous monitoring of LDL autooxidation, especially for the in vitro testing of the protective effect of antioxidant compounds.     

Continuous Monitoring of in Vztro Oxidation of Human Low Density Lipoprotein

The kinetics of the oxidation of human low densit) lipoprotein (LDL) can be measured continuously by monitoring the change of the 234 nm diene absorption. The time-course shows three consecutive phases, a lag-phase during which the diene absorption increases only weakly. a propagation phase with a rapid increase of the diene absorption and finally a decomposition phase. The increase of the dienes is highly correlated with the increase of MDA or lipid hydroperoxides. The duration of the lag-phase is determined by the endogenous antioxidants contained in LDL (vitamin E. carotenoids. retinylstearate). Water-soluble antioxidants (ascorbic acid. urate) added in micromolar concentrations prolong the lag-phase in a concentration-dependent manner. The determination of the lag-phase is a convenient and objective procedure for determining the susceptibility of LDL from different donors towards oxidation as well as effects of pro-and antioxidants.     

Generation in Human Plasma of Misfolded, Aggregation-Prone Electronegative Low Density Lipoprotein

Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(−), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(−) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(−) during incubation of unprocessed human plasma at 37°C. In addition to a higher fraction of amyloidogenic LDL(−), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoprotein's structure, which misfolds and becomes aggregation-prone.     

人可溶性低密度脂蛋白受体在甲醇酵母中的表达

为获得低密度脂蛋白受体配基结合结构域在甲醇酵母中的分泌表达 ,首先用RT PCR方法以人肝癌Bel 740 2总RNA为模板扩增了编码低密度脂蛋白受体配基结合结构域的基因片段。核酸测序分析表明克隆到的DNA片段的序列与报道的人LDLR的cDNA序列相同。然后构建了甲醇酵母表达质粒pPIC9K sLDLr ,并将其线性化后用电穿孔法导入PichiapastorisGS115。分别用SDS PAGE、Westernblot和Ligandbindingblot对GS115 pPIC9K sLDLr上清中的重组sLDLR进行鉴定。SDS PAGE和Westernblot分析表明表达的sLDLR的表观分子量为 36kD。Ligandbindingblot分析表明表达的sLDLR具有配基结合的生物学活性     

Mathematical Model for Low Density Lipoprotein (LDL) Endocytosis by Hepatocytes

Individuals with elevated levels of plasma low density lipoprotein (LDL) cholesterol (LDL-C) are considered to be at risk of developing coronary heart disease. LDL particles are removed from the blood by a process known as receptor-mediated endocytosis, which occurs mainly in the liver. A series of classical experiments delineated the major steps in the endocytotic process; apolipoprotein B-100 present on LDL particles binds to a specific receptor (LDL receptor, LDL-R) in specialized areas of the cell surface called clathrin-coated pits. The pit comprising the LDL–LDL-R complex is internalized forming a cytoplasmic endosome. Fusion of the endosome with a lysosome leads to degradation of the LDL into its constituent parts (that is, cholesterol, fatty acids, and amino acids), which are released for reuse by the cell, or are excreted. In this paper, we formulate a mathematical model of LDL endocytosis, consisting of a system of ordinary differential equations. We validate our model against existing in vitro experimental data, and we use it to explore differences in system behavior when a single bolus of extracellular LDL is supplied to cells, compared to when a continuous supply of LDL particles is available. Whereas the former situation is common to in vitro experimental systems, the latter better reflects the in vivo situation. We use asymptotic analysis and numerical simulations to study the longtime behavior of model solutions. The implications of model-derived insights for experimental design are discussed.