Napoli C, Triggiani M, Palumbo G, Condorelli M, Chiariello M, Ambrosio G
Division of Clinical Immunology, Federico II University of Naples, Italy.
Basic Res Cardiol. 1997 Apr;92(2):96-105. doi: 10.1007/BF00805570.
Posttranslational nonenzymatic glycosylation of native low-density lipoprotein (n-LDL) occurs both in vitro and in vivo in diabetic patients. Glycosylated LDL (glc-LDL) behave similarly to oxidized LDL in some respects. In fact, unlike n-LDL, uptake the glc-LDL can occur in part by the "scavenger" receptor(s), as also demonstrated for oxidized LDL. The enzyme acetylhydrolase, carried by LDL, catabolizes platelet activating factor (PAF). This enzymatic activity is inhibited in oxidized LDL. However, it is unknown whether glc-LDL have reduced acetylhydrolase activity.
The first aim of the study was to investigate whether glc-LDL were more susceptible than n-LDL to oxidative modification, and which different oxygen radical species were involved in the phenomenon. Moreover, in order to investigate whether glycosylation may affect acetylhydrolase, we also measured this enzymatic activity in both n- and glc-LDL.
In vitro glc-LDL and n-LDL were exposed to the oxidants xanthine/xanthine oxidase (X/XO; 2 mM and 100 mU/ml, respectively), or CuSO4 (10 microM) for 18 hs at 37 degrees C. Parallel experiments were done in the presence of the superoxide radical scavenger superoxide dismutase (SOD; 330 U/ml), the hydrogen peroxide scavenger catalase (1000 U/ml), or the hydroxyl radical scavenger dimethylthiourea (10 mM) or dimethylsulfoxide (1 mM). Standards of PAF and lyso-PAF were visualized with iodine vapors after separation by thin layer chromatography. The distribution of label was determined by an imaging scanner. Labeled products were then isolated from the chromatography plate, and the amount of 3H-lyso-PAF formed was determined by liquid scintillation counting.
Glc-LDL were more susceptible than n-LDL to lipid peroxidation (n-LDL 22.9 +/- 3.4 vs 34.8 +/- 4.2* nmoles/MDA/mg of protein in glc-LDL oxidized by X/XO and n-LDL 28.9 +/- 4.2 vs 40.4 +/- 4.1* in glc-LDL oxidized by CuSO4, *p < 0.05 vs n-LDL). SOD, but not other scavengers, prevented peroxidation, indicating an obligatory role for superoxide radicals. Oxidation of glc-LDL also induced a higher degree of apolipoprotein-B100 modifications than n-LDL, with increased electrophoresis mobility and decreased TNBS reactivity. These effects were similarly prevented by SOD. Finally, acetylhydrolase activity was significantly lower in glc-LDL than in n-LDL.
Glycosylation increases LDL oxidation due to superoxide radicals, and also reduces acetylhydrolase activity. These phenomenona may contribute to enhance and/or accelerate the progression of atherosclerosis in diabetic patients.
天然低密度脂蛋白（n-LDL）的翻译后非酶糖基化在糖尿病患者体内外均可发生。糖基化低密度脂蛋白（glc-LDL）在某些方面的表现与氧化型低密度脂蛋白相似。事实上，与n-LDL不同，glc-LDL可部分通过“清道夫”受体摄取，氧化型低密度脂蛋白也是如此。LDL携带的乙酰水解酶可分解血小板活化因子（PAF）。这种酶活性在氧化型低密度脂蛋白中受到抑制。然而，尚不清楚glc-LDL的乙酰水解酶活性是否降低。
本研究的首要目的是探究glc-LDL是否比n-LDL更容易发生氧化修饰，以及该现象涉及哪些不同的氧自由基。此外，为了研究糖基化是否会影响乙酰水解酶，我们还测量了n-LDL和glc-LDL中的这种酶活性。
体外将glc-LDL和n-LDL分别在37℃下暴露于氧化剂黄嘌呤/黄嘌呤氧化酶（X/XO；分别为2 mM和100 mU/ml）或硫酸铜（10 μM）中18小时。在超氧阴离子清除剂超氧化物歧化酶（SOD；330 U/ml）、过氧化氢清除剂过氧化氢酶（1000 U/ml）、羟基自由基清除剂二甲基硫脲（10 mM）或二甲基亚砜（1 mM）存在的情况下进行平行实验。PAF和溶血PAF标准品经薄层色谱分离后用碘蒸气显色。通过成像扫描仪测定标记物的分布。然后从色谱板上分离出标记产物，通过液体闪烁计数法测定形成的3H-溶血PAF的量。
Glc-LDL比n-LDL更容易发生脂质过氧化（经X/XO氧化的glc-LDL中n-LDL为22.9±3.4 vs 34.8±4.2* nmoles/MDA/mg蛋白，经硫酸铜氧化的glc-LDL中n-LDL为28.9±4.2 vs 40.4±4.1*，*与n-LDL相比，p<0.05）。SOD可阻止过氧化，而其他清除剂则不能，这表明超氧阴离子自由基起关键作用。glc-LDL的氧化还比n-LDL诱导了更高程度的载脂蛋白B100修饰，电泳迁移率增加，TNBS反应性降低。SOD同样可阻止这些效应。最后，glc-LDL中的乙酰水解酶活性显著低于n-LDL。
糖基化由于超氧阴离子自由基增加了LDL氧化，并且还降低了乙酰水解酶活性。这些现象可能有助于增强和/或加速糖尿病患者动脉粥样硬化的进展。
