Abstract—

The elimination kinetics of carbonyl-modified low density lipoproteins (LDL) from rabbit bloodstream was studied using isolated LDL of rabbits and humans after preliminary biotinylation or labeling with FITC. Rabbit or human blood plasma LDL were isolated using differential ultracentrifugation in a density gradient; after labeling by biotinylation or by FITC, LDL were modified with various low molecular weight natural dicarbonyls: malondialdehyde (MDA), glyoxal or methylglyoxal. Native (control) and dicarbonyl-modified biotinylated or FITC-labeled LDL were injected into the ear vein of rabbits, and blood samples were taken at certain time intervals. The content of biotinylated LDL in blood plasma was determined by an enzyme immunoassay method; FITC-labeled LDL was determined from the fluorescence spectra. It has been found that glyoxal- and methylglyoxal-modified rabbit and human LDL circulate in the bloodstream of rabbits for almost the same period as native (unmodified) LDL. In contrast to this, MDA-modified rabbit and human LDL were very quickly eliminated from the rabbit bloodstream. Dicarbonyl-modified LDL from human blood plasma is not associated with red blood cells or endothelial cells. We found that using the Oxidized LDL ELISA kits (Mercodia, Sweden) it was possible to identify mainly MDA-modified LDL. The level of MDA-modified LDL in the blood plasma of CHD patients sharply decreased during therapy with evolocumab, the hypocholesterolemic inhibitor of PCSK₉ (proprotein convertase of subtilisin/kexin type 9), which activates LDL reutilization in the liver cells. These results explain the extremely rapid clearance of MDA-modified LDL in our experiments by their increased utilization in hepatocytes. The results obtained indicate a high atherogenicity of glyoxal- and methylglyoxal-modified LDL, long-term circulating in the bloodstream.

中文翻译：

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羰基修饰的低密度脂蛋白在兔体内的清除

摘要—

在初步生物素化或用FITC标记后，使用分离的兔和人LDL研究了兔血中羰基修饰的低密度脂蛋白（LDL）的消除动力学。使用差速超速离心法以密度梯度分离兔或人血浆中的LDL。通过生物素化或FITC标记后，LDL被各种低分子量天然二羰基化合物修饰：丙二醛（MDA），乙二醛或甲基乙二醛。将天然的（对照）和二羰基修饰的生物素化或FITC标记的LDL注入兔的耳静脉，并在一定时间间隔采集血样。用酶免疫法测定血浆中生物素化低密度脂蛋白的含量。从荧光光谱确定FITC标记的LDL。已经发现，乙二醛和甲基乙二醛修饰的兔和人LDL在兔的血液中循环的时间与天然（未修饰的）LDL几乎相同。与此相反，MDA修饰的兔和人LDL很快从兔血流中清除。来自人血浆的二羰基修饰的LDL与红细胞或内皮细胞无关。我们发现使用氧化的LDL ELISA试剂盒（Mercodia，瑞典）可以鉴定出主要是MDA修饰的LDL。在使用PCSK降胆固醇抑制剂evolocumab治疗期间，CHD患者血浆中MDA修饰的LDL水平急剧下降 MDA修饰的兔和人LDL很快从兔血中消除。来自人血浆的二羰基修饰的LDL与红细胞或内皮细胞无关。我们发现使用氧化的LDL ELISA试剂盒（Mercodia，瑞典）可以鉴定出主要是MDA修饰的LDL。在使用PCSK降胆固醇抑制剂evolocumab治疗期间，CHD患者血浆中MDA修饰的LDL水平急剧下降 MDA修饰的兔和人LDL很快从兔血中消除。来自人血浆的二羰基修饰的LDL与红细胞或内皮细胞无关。我们发现使用氧化的LDL ELISA试剂盒（Mercodia，瑞典）可以鉴定出主要是MDA修饰的LDL。在使用PCSK降胆固醇抑制剂evolocumab治疗期间，CHD患者血浆中MDA修饰的LDL水平急剧下降₉（枯草杆菌蛋白酶/ kexin型9的原蛋白转化酶），可激活肝细胞中LDL的再利用。这些结果解释了在我们的实验中，由于MDA修饰的LDL在肝细胞中的利用率提高，因此可以极其迅速地清除它们。获得的结果表明乙二醛和甲基乙二醛改性的LDL具有很高的动脉粥样硬化性，可以在血液中长期循环。