Anion Exchange HPLC Isolation of High-Density Lipoprotein (HDL) and On-Line Estimation of Proinflammatory HDL
Anion Exchange HPLC Isolation of High-Density Lipoprotein (HDL) and On-Line Estimation of Proinflammatory HDL
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DOI：
10.1371/journal.pone.0091089
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发表时间：
2014-03-07
期刊：
影响因子：
3.7
通讯作者：
Pritchard, Kirkwood A., Jr.
中科院分区：
文献类型：
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作者：
Ji, Xiang;Xu, Hao;Pritchard, Kirkwood A., Jr.
前炎性高密度脂蛋白(p-HDL)是心血管疾病的生物标志物。镰状细胞病(SCD)的特征是慢性氧化应激状态，许多人认为这种状态在形成p-高密度脂蛋白中发挥了作用。为了测定p-高密度脂蛋白，沉淀了含有载脂蛋白的载脂蛋白B。上清的高密度脂蛋白与氧化剂/低密度脂蛋白或单独的氧化剂孵育，并用二氯荧光素(DCFH)监测高密度脂蛋白的氧化速率。虽然apoB沉淀有利于高密度脂蛋白的分离，但产生的上清液基质可能会影响高密度脂蛋白的氧化。为了确定上清液对p-高密度脂蛋白测定的影响，我们用阴离子交换层析(AE)从SCD患者血浆中纯化了高密度脂蛋白，并测定了其相对于上清液的氧化速率。SCD降低总胆固醇，但不降低甘油三酯或高密度脂蛋白，并增加游离细胞(Cf)、血红蛋白(Hb)和黄嘌呤氧化酶(XO)。用AE-HPLC分离的高密度脂蛋白经apoB沉淀后，其上清液中的p-高密度脂蛋白水平低于高密度脂蛋白。XO+黄嘌呤(X)和cf Hb促进纯化的高密度脂蛋白氧化。尽管平板法和AE-HPLC法都表明SCD血浆中的p-HDL值与cf-Hb直接相关，但平板法得到的p-HDL值受cf-Hb的影响比AE-HPLC法产生的p-HDL值受cf-Hb影响更大。AE-HPLCp-HDLE测定减少了上清液的影响，表明SCD增加了p-HDL值。
Proinflammatory high-density lipoprotein (p-HDL) is a biomarker of cardiovascular disease. Sickle cell disease (SCD) is characterized by chronic states of oxidative stress that many consider to play a role in forming p-HDL. To measure p-HDL, apolipoprotein (apo) B containing lipoproteins are precipitated. Supernatant HDL is incubated with an oxidant/LDL or an oxidant alone and rates of HDL oxidation monitored with dichlorofluorescein (DCFH). Although apoB precipitation is convenient for isolating HDL, the resulting supernatant matrix likely influences HDL oxidation. To determine effects of supernatants on p-HDL measurements we purified HDL from plasma from SCD subjects by anion exchange (AE) chromatography, determined its rate of oxidation relative to supernatant HDL. SCD decreased total cholesterol but not triglycerides or HDL and increased cell-free (cf) hemoglobin (Hb) and xanthine oxidase (XO). HDL isolated by AE-HPLC had lower p-HDL levels than HDL in supernatants after apoB precipitation. XO+xanthine (X) and cf Hb accelerated purified HDL oxidation. Although the plate and AE-HPLC assays both showed p-HDL directly correlated with cf-Hb in SCD plasma, the plate assay yielded p-HDL data that was influenced more by cf-Hb than AE-HPLC generated p-HDL data. The AE-HPLC p-HDL assay reduces the influence of the supernatants and shows that SCD increases p-HDL.