I型胶原对低密度脂蛋白氧化反应敏感性的影响

在胶原存在条件下低密度脂蛋白（ＬＤＬ）的氧化反应性的改变一直未见报道,通过在体外分离大鼠Ｉ型胶原并制成胶原凝胶,发现铜离子（Ｃｕ＾２＋）介导的ＬＤＬ氧化反应的潜伏期明显延长,所生成的硫代巴比妥酸反应物（ＴＢＡＲＳ）最大值远低于对照组,胶原组氧化ＬＤＬ（ｏｘ－ＬＤＬ）的相对电泳迁移率（ＲＥＭ）也较对照组为低;在偶氮类化合物２,２＇－盐酸脒基丙烷（ＡＡＰＨ）诱导的ＬＤＬ氧化过程中,胶原组与对照组的ＬＤ     

芦丁和槲皮素对低密度脂蛋白氧化修饰的抑制作用

以低密度脂蛋白（ＬＤＬ）氧化修饰为模型和以硫代巴比妥酸反应物质（ＴＢＡＳ）生成量以及ＬＤＬ的α－Ｔｏｃｏｐｈｅｒｏｌ和荧光物质含量为指标,以时间效应和浓度效应说明槲皮素和芦丁能明显地抑制Ｃａ＾２＋诱导的ＬＤＬ氧化修饰但其抗氧化修饰的程度无明显差异。它们对已受到Ｃｕ＾２＋氧化修饰的ＬＤＬ的过氧化无明显地终止作用。     

I型胶原对巨噬细胞摄取氧化低密度脂蛋白的作用

为探讨胶原的存在对细胞摄取氧化低密度脂蛋白（ｏｘ－ＬＤＬ）的影响,本研究在体外制成Ｉ型胶原凝胶和巨噬细胞实验体系,ＬＤＬ经Ｃｕ＾２＋催化氧化,丙二醛（ＭＤＡ）及乙酰化修饰后,与胶原的结合能力明显增强,但４－羟基壬烯醛（ＨＮＥ）修饰的ＬＤＬ与胶原的结合能力反应不如天然ＬＤＬ。当小鼠腹腔巨噬细胞培养在胶原凝胶上时,其对ｏｘ－ＬＤＬ的摄取明显减少,这时大部分ｏｘ－ＬＤＬ为胶原凝胶所结合,如用细胞松弛素Ｄ     

利用高山红景天培养细胞生物转化外源酪醇生产红景天甙的研究

高山红景天（ＲｈｏｄｉｏｌａｓａｃｈａｌｉｎｅｎｓｉｓＡ．Ｂｏｒ．）培养细胞中,甙元酪醇在细胞生长静止期大量积累,而此时糖基化反应的效率很低,因而红景天甙（ｓａｌｉｄｒｏｓｉｄｅ）产量较低。考虑到培养细胞中酪醇葡萄糖基转移酶的活性在指数生长期达到最高,考察了在指数生长期添加外源酪醇生物转化生产红景天甙的可能性,并探讨了酪醇添加浓度、添加方法及细胞密度对酪醇转化率及红景天甙产量的影响。结果表明,细胞在酪醇浓度为１ｍｍｏｌ／Ｌ的培养基中培养２４ｈ后可使酪醇转化率达到９５％;过高的酪醇浓度（＞３ｍｍｏｌ／Ｌ）对细胞生长及酪醇转化率都有明显抑制作用;通过较低浓度酪醇的３次重复添加,可使细胞密度为６ｇＤＷ／Ｌ、１２ｇＤＷ／Ｌ及１８ｇＤＷ／Ｌ的培养物中的红景天甙产量分别达到１３２０ｍｇ／Ｌ、１７４０ｍｇ／Ｌ和１９８０ｍｇ／Ｌ。     

氧化LDL的不同组分在抑制巨噬细胞释放NO中的作用

研究表明：氧化ＬＤＬ（Ｏｘ－ＬＤＬ）可抑制ＬＰＳ诱导的巨噬细胞ＮＯ释放,而正常ＬＤＬ和乙酰化ＬＤＬ则无抑制作用。用谷胱甘肽地的酶模拟物ｅｂｓｅｌｅｎ清除Ｏｘ－ＬＤＬ上的脂氢过氧化物对其抑制作用没有影响。Ｏｘ－ＬＤＬ的蛋白组分对ＮＯ释放也没有影响,而脂质组分则有抑制作用。ＬＤＬ脂质的主要成分亚油酸和卵磷脂无论单独还是共同氧化后对ＮＯ释放都没有影响,而亚油酸和胆固醇一起氧化后则对ＮＯ的释放有很强的抑制     

氧化低密度脂蛋白

1 .氧化低密度脂蛋白 (ＬＤＬ)的发现形成动脉粥样硬化过程中的泡沫细胞病变与巨噬细胞的脂化胆固醇积累相关 ,在巨噬细胞极少发现ＬＤＬ受体 ,特别是遗传性ＬＤＬ受体缺乏的同形合子患者的家族性高胆固醇血症及其动物模型中 ,极早出现动脉粥样硬化病变。以后用抗氧化药能阻止高ＬＤＬ血症的实验兔发生动脉粥样硬化。用特异性的氧化ＬＤＬ的单克隆抗体 ,采用免疫组织化学法 ,从高脂血症的患者及动物模型的动脉粥样硬化病变部位检出氧化ＬＤＬ。ＬＤＬ的氧化变性的实际部位在活体内 ,特别是在血管壁内发生 ,对动脉粥样硬化的形成起重要作用。…     

氧化修饰低密度脂蛋白与动脉粥样硬化

动脉粥样硬化的发生发展与低密度脂蛋白受到氧化修饰有关。本文从以下四个方面对本室的工作进行了综述：（１）动脉粥样硬化机体受到脂质过氧化损伤;（２）Ｏｘ－ＬＤＬ对内皮细胞、平滑肌细胞和巨噬细胞的毒性效应;（３）Ｏｘ－ＬＤＬ和ＭＤＡ－ＬＤＬ的比较及与Ｏｘ－ＬＤＬ和ＭＤＡ－ＬＤＬ结合的清道夫受体的特征;（４）不同方法对ＬＤＬ氧化修饰的比较和以ＬＤＬ氧化修饰为模型对某些物质的抗氧化修饰研究。研究结果为动脉粥     

高密度脂蛋白体外氧化修饰动力学研究

在体外ＨＤＬ在Ｃｕ＾２＋诱导下可发生氧化修饰,为了探讨体外务浆高密度脂蛋白（ＨＤＬ）氧化修饰中几种产物的动力学改变,用Ｃｕ＾２＋与ＨＤＬ保温２￣２４ｈ,分别观察了ＨＤＬ氧化修饰过程中硫代巴比妥酸反应物质（ＴＢＡＲＳ）,脂氢过氧化物（ＬＯＯＨ）、共轭二烯（ＣＤ）及相对电泳迁移率（ＲＥＭ）等的变化。结果显示,ＬＯＯＨ和ＣＤ两个指标动力学变化相似,呈现延滞期,扩增期和下降期三个时相,而ＴＢＡＲＳ和ＲＥＭ     

基因工程菌高密度发酵液中碳源物质甘油的定量检测及其浓度的优化控制

比较了高碘酸氧化法,铜离子络合法、高压液相色谱法和甘油激酶法四种不同的方法定量测定重组工程菌ＹＫ５３７／ＰＳＢ－ＴＫ高密度发酵液中甘油的浓度,发现甘油激酶法可以排除发酵液中其他物质的干扰,精确测定甘油的含量。在此基础上对发酵培养中甘油的浓度进行了优化,结果表明,甘油浓度控制在较低的水平有利于菌体密度的提高。在５Ｌ自动发酵罐中,控制甘油浓度在５ｇ／Ｌ左右,经２０ｈ的培养,最终细菌密度达到１２０ＯＤ６     

丙二醛和Cu~（2＋）对低密度脂蛋白修饰的比较研究

实验比较了丙二醛修饰的低密度脂蛋白（ＭＤＡ－ＬＤＬ）和氧化修饰的低密度脂蛋白（ｏ－ＬＤＬ）的某些理化性质;用放射性受体分析法,从标记配体的可饱和性、可逆性、高亲和力和立体选择性等几方面,证实在小鼠腹腔巨噬细胞（ＭＰＭ）表面存在有特异的ＭＤＡ－ＬＤＬ受体,获得了一系列能反应受体特征的参数;同时,还将ＭＤＡ－ＬＤＬ受体的特征和ｏ－ＬＤＬ的受体特征进行了比较。这些结果将有助于进一步研究ＭＰＭ清道夫受体的多态性及其与动脉粥样硬化的发病原理和防治的关系。     

Antioxidants and resistance against oxidation of porcine LDL subfractions

The objective of this study was to determine the level of antioxidants, the content of fatty acids and peroxidation products, and the resistance against oxidation of native porcine LDL1 and LDL2. There were no significant differences in the fatty acid distribution of both native low density lipoprotein (LDL) subfractions, which was similar to that of human LDL. The total amount of alpha- and gamma-tocopherol of pig LDL was significantly lower than in human LDL, and beta-carotene, lycopene, and retinyl esters were totally absent. Levels of thiobarbituric acid-reacting substances (TBARS) and lipid peroxides in freshly isolated pig LDL subfractions were below or only slightly above the detection limit. The susceptibility to oxidation of both LDL subfractions was investigated by addition of Cu2+ as prooxidant. The results show that pig LDL subfractions are much more susceptible to oxidation as measured by the duration of the lag phase preceding the onset of rapid lipid peroxidation. From the low content of vitamin E one would expect even much shorter lag phases. The possibility therefore exists that pig LDL contains additional, and as yet unidentified, antioxidants.     

Guinea pig low density lipoproteins: structural and metabolic heterogeneity

The structural and metabolic heterogeneity of low density lipoproteins (LDL, d 1.024-1.100 g/ml) has been investigated in the guinea pig. Two LDL subfractions, of d 1.024-1.050 and 1.050-1.100 g/ml, respectively, were isolated by sequential ultracentrifugation; while both were enriched in cholesteryl ester and apoB-100, the former was heterogeneous displaying three particle size species of diameters 26.9, 25.6, and 24.7 nm, whereas the denser subfraction was relatively homogeneous containing a single, smaller species (diam. 23.6 nm). The fractional catabolic rates (FCR) of the two LDL subfractions were alike (approximately 0.090 pools/hr) in the guinea pig in vivo. After modification of each subfraction by reductive methylation, the FCRs were reduced similarly and indicated that 70-80% of degradation occurred via the cellular LDL receptor pathway. However, the intravascular metabolism of these LDL subfractions, determined from the radioactive content of density gradient fractions as a function of time after injection of radiolabeled native or chemically modified LDL, tended to be distinct. Thus, while radiolabeled apoB-100 in the lighter subfraction maintained the initial density profile up to 48 hr, the radioactive profile of its methylated counterpart changed, the proportion of radioactivity in the lighter gradient fractions (d 1.027-1.032 g/ml) increasing while that in the denser (d 1.037-1.042 g/ml) fractions diminished. A more marked transformation occurred in LDL of d 1.050-1.100 g/ml, in which the radioactive profile shifted towards lighter particles of the d 1.024-1.050 g/ml species; this shift was partially dependent on the LDL receptor, since it was more pronounced in the methylated subfraction. Furthermore, a net increase in the radioactive content of gradient subfractions 7 to 9 (d 1.032-1.042 g/ml) was found 10 hr after injection of methylated LDL of d 1.050-1.100 g/ml, at which time the bulk of LDL radioactivity had been removed from plasma. Several mechanisms, acting alone or in combination, may account for these findings; among them, some degree of transformation of dense to lighter LDL species appears a prerequisite. In conclusion, our data attest to the structural heterogeneity of circulating LDL in the guinea pig, and suggest that the intravascular processing and metabolism of LDL particle subspecies is directly related to their structure and physicochemical properties.     

Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.

Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized.The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.     

Phospholipase A(2) modification of low density lipoproteins forms small high density particles with increased affinity for proteoglycans and glycosaminoglycans.

The presence of a lipoprotein profile with abundance of small, dense low density lipoproteins (LDL), low levels of high density lipoproteins (HDL), and elevated levels of triglyceride-rich very low density lipoproteins is associated with an increased risk for coronary heart disease. The atherogenicity of small, dense LDL is believed to be one of the main reasons for this association. This particle contains less phospholipids (PL) and unesterified cholesterol than large LDL, and the apoB-100 appears to occupy a more extensive area at its surface. Although there are experiments that suggest a metabolic pathway leading to the overproduction of small, dense LDL, no clear molecular model exists to explain its association with atherogenesis. A current hypothesis is that small, dense LDL, because of its higher affinity for proteoglycans, is entrapped in the intima extracellular matrix and is more susceptible to oxidative modifications than large LDL. Here we describe how a specific reduction of approximately 50% of the PL of a normal buoyant LDL by immobilized phospholipase A(2) (PLA(2)) (EC 3.1.1.4) produces smaller and denser particles without inducing significant lipoprotein aggregation (<5%). These smaller LDL particles display a higher tendency to form nonsoluble complexes with proteoglycans and glycosaminoglycans than the parent LDL. Binding parameters of LDL and glycosaminoglycans and proteoglycans produced by human arterial smooth muscle cells were measured at near to physiological conditions. The PLA(2)-modified LDL has about 2 times higher affinity for the sulfated polysaccharides than control LDL. In addition, incubation of human plasma in the presence of PLA(2) generated smaller LDL and HDL particles compared with the control plasma incubated without PLA(2). These in vitro results indicate that the reduction of surface PL characteristic of small, dense LDL subfractions, besides contributing to its small size and density, may enhance its tendency to be retained by proteoglycans.     

Evidence for heterogeneity of low-density lipoprotein metabolism in the cynomolgus monkey

Preliminary studies were performed to establish whether there was kinetic heterogeneity in the metabolism of subclasses of low-density lipoproteins (LDL) in the cynomolgus monkey. Previous studies of the effects of inhibition of hepatic triglyceride lipase in this species had shown an increase in the mass of lighter LDL (Sf greater than 9) and a decrease in the mass of denser LDL. LDL (1.019 less than d less than 1.063) were subdivided into two subfractions LDL1 (1.019 less than d less than 1.035) and LDL2 (1.035 less than d less than 1.063) by ultracentrifugation. The lipoproteins in these two fractions could be shown to have different flotation by analytic and isopycnic ultracentrifugation. When tracer amounts of homologous 125I-labeled very-low-density lipoproteins (VLDL) were injected into chow-fed cynomolgus monkeys, apoB radioactivity appeared in LDL1 prior to its appearance in LDL2. [125I]LDL1 injected into the monkey was removed from the LDL1 density subclass with a half-life of 5.5-10.3 h. Much of the radioactivity injected as LDL1 was converted to denser LDL (LDL2). Labeled LDL2 injected into the monkey was not converted to LDL1. Thus, at least two kinetically distinct subpopulations of LDL circulate in the plasma of this species. The lighter LDL is to a large extent a metabolic precursor of the more dense LDL (LDL2).     

Rapid isolation of VLDL subfractions: assessment of composition and susceptibility to copper-mediated oxidation

VLDLs, synthesized and released by the liver, are a heterogeneous group of particles of varying composition and metabolic fates. A method is described for the rapid isolation of VLDL into four subfractions (A-D) and assessment of their susceptibility to oxidation. The total isolation procedure required less than 3.5 h, and was achieved by gradient ultracentrifugation. Each subfraction was assessed for triglyceride, cholesterol, and apolipoprotein B (apoB) composition and for the presence of contaminants such as albumin and urate. The oxidation potential, in the presence of copper ions, of each subfraction was also assessed. This rapid procedure produced VLDL fractions analogous to those produced by a previously reported but more prolonged isolation method. Comparison of the two procedures demonstrated that lipid and apoB were similar, while the rapid procedure produced subfractions void of albumin and urate contamination and lower in preformed hydroperoxides. Compositional changes were found between the subfractions: as the subfractions became smaller and more dense (A-->D), there was a decrease in the ratio of triglyceride to apoB and an increase in the ratio of cholesterol to apoB, also arachidonic acid was increased in subfraction D compared with subfractions A, B, and C. The smaller subfractions were more susceptible to oxidation, a trend similar to that reported previously for the oxidation of LDL subfractions.     

Oxidation-labile subfraction of human plasma low density lipoprotein isolated by ion-exchange chromatography

We isolated subfractions of human plasma low density lipoprotein (LDL) using ion-exchange chromatography. Plasma LDL from normolipidemic subjects were applied to a DEAE Sepharose 6B column. After elution of the bulk of LDL at 150 mM NaCl (the major fraction), the residual LDL was eluted at 500 mM NaCl and designated as the minor fraction. The minor fraction, only less than 1% of total LDL, tended to be somewhat similar in certain properties to oxidized LDL, e.g., an increased negative charge, higher protein/cholesterol ratio, and a higher flotation density than native LDL. These results were consistent with data reported by Avogaro et al. (1988. Arteriosclerosis. 8: 79-87). However, assays of 125I-labeled LDL binding activity for LDL receptors equal to that of the major fraction. Incorporation of [14C]oleate into cholesteryl ester [acyl-CoA:cholesterol acyltransferase (ACAT) activity] in mouse peritoneal macrophages incubated with the minor fraction was only slightly greater than that with the major fraction. Incubation of the minor fraction with 0.5 microM Cu2+ caused a remarkable stimulation of ACAT activity, while stimulation by the major fraction required incubation with 5 microM Cu2+, suggesting that the minor fraction was relatively labile to oxidation. The minor but definite presence of a plasma LDL subfraction more negative and susceptible to oxidation implicates the possibility of its association with atherogenesis.     

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1

Apolipoprotein A-I (apoA-I) and an apoA-I peptide mimetic removed seeding molecules from human low density lipoprotein (LDL) and rendered the LDL resistant to oxidation by human artery wall cells. The apoA-I-associated seeding molecules included hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE). LDL from mice genetically susceptible to fatty streak lesion formation was highly susceptible to oxidation by artery wall cells and was rendered resistant to oxidation after incubation with apoA-I in vitro. Injection of apoA-I (but not apoA-II or murine serum albumin) into mice rendered their LDL resistant to oxidation within 3 h. Infusion of apoA-I into humans rendered their LDL resistant to oxidation within 6 h.We conclude that 1) oxidation of LDL by artery wall cells requires seeding molecules that include HPODE and HPETE; 2) LDL from mice genetically susceptible to atherogenesis is more readily oxidized by artery wall cells; and 3) normal HDL and its components can remove or inhibit the activity of lipids in freshly isolated LDL that are required for oxidation by human artery wall cells.     

The fatty acid distribution in low density lipoprotein in diabetes.

Atherosclerosis is commonly found in diabetes. There is an association between small dense low density lipoprotein (LDL) phenotype, which is more prevalent in the diabetic state, and atherosclerosis. Small dense LDL is more easily oxidised and it is possible that fatty acid compositional changes, particularly an increase in polyunsaturated fatty acids, could underlie this association. However, there is little information about fatty acids in the different LDL phenotypes in the literature. This study examined LDL subfraction composition in 18 non-insulin-dependent diabetic (NIDDM) patients and 11 control subjects. LDL was isolated and fractionated into LDL 1, 2 and 3 by density gradient ultracentrifugation. NIDDM patients had significantly more fatty acids in all LDL subfractions than control subjects (P<0.01). Palmitic and linoleic acid were significantly greater in all subfractions in the diabetic patients compared to control subjects (P<0.01) and palmitoleic and oleic acids were also greater in LDL1 and LDL2 in diabetic patients (P<0.01). We conclude that in NIDDM fatty acids are increased in all LDL subfractions and this may be the reason for the increased atherosclerosis in diabetes irrespective of phenotype.     

The fatty acid distribution in low density lipoprotein in diabetes

Atherosclerosis is commonly found in diabetes. There is an association between small dense low density lipoprotein (LDL) phenotype, which is more prevalent in the diabetic state, and atherosclerosis. Small dense LDL is more easily oxidised and it is possible that fatty acid compositional changes, particularly an increase in polyunsaturated fatty acids, could underlie this association. However, there is little information about fatty acids in the different LDL phenotypes in the literature. This study examined LDL subfraction composition in 18 non-insulin-dependent diabetic (NIDDM) patients and 11 control subjects. LDL was isolated and fractionated into LDL 1, 2 and 3 by density gradient ultracentrifugation. NIDDM patients had significantly more fatty acids in all LDL subfractions than control subjects (P<0.01). Palmitic and linoleic acid were significantly greater in all subfractions in the diabetic patients compared to control subjects (P<0.01) and palmitoleic and oleic acids were also greater in LDL1 and LDL2 in diabetic patients (P<0.01). We conclude that in NIDDM fatty acids are increased in all LDL subfractions and this may be the reason for the increased atherosclerosis in diabetes irrespective of phenotype.