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Introduction
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Immunology and Infection

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Published: October 12, 2017
doi:
10.3791/56325
, , , , , ,
1University of California, Los Angeles, 2Centre for Biomedical Research,Burnet Institute, 3School of Health and Biomedical Sciences,RMIT University, 4Department of Infectious Diseases,Monash University

Summary

我们在这里描述了一种荧光无细胞生化测定高密度脂蛋白脂质过氧化的方法。这种快速和重现性的分析可以用来确定高密度脂蛋白功能的大规模研究, 并能有助于我们的理解 hdl 功能的人类疾病。

Abstract

低高密度脂蛋白胆固醇 (HDL C) 水平是动脉粥样硬化性心血管疾病 (CVD) 最强大的独立阴性预测因子之一。hdl 的结构和功能, 而不是高密度脂蛋白 C 可以更准确地预测动脉粥样硬化。一些高密度脂蛋白蛋白和脂质成分的改变会损害 hdl 的功能, 如动脉粥样硬化等炎症状态。HDL 的功能通常是由细胞基础的化验, 如胆固醇外流化验, 但这些化验有许多缺点缺乏标准化。与细胞检测相比, 无单元检测可能给 HDL 功能提供更有力的措施。高密度脂蛋白氧化损害 hdl 功能。高密度脂蛋白在脂质过氧化物转运中有重要作用, 高脂质过氧化物与 hdl 功能异常有关。在解释酶荧光法测定脂质氧化状态的结果时, 应考虑脂探针的相互作用。这促使我们开发一种无细胞生物化学酶方法来评估 hdl 脂质过氧化物含量 (HDLox), 从而导致高密度脂蛋白功能障碍。这种方法是基于酶辣根酶 (HRP) 和荧光 Amplex 红, 可以量化 (不含胆固醇氧化酶) 的脂质过氧化物含量每毫克的高密度脂蛋白 C。这里的一个协议是 describedfor 测定 HDL 脂质过氧化荧光试剂。实验条件的严格标准化可以降低测定的变异性。较高的 HDLox 值与降低的 HDL 抗氧化功能相关。该方法的读数与经验证的细胞检测的读数, 心血管疾病的替代措施, 全身炎症, 免疫功能障碍, 以及相关的心血管和代谢风险表型有关。这种技术方法是一种强有力的方法来评估 HDL 功能在人类疾病中, 全身炎症, 氧化应激和氧化脂有一个关键的作用 (如动脉粥样硬化)。

Introduction

动脉粥样硬化性心血管疾病 (CVD) 是导致全球死亡的主要原因1,2。流行病学研究表明, 低浓度高密度脂蛋白 (HDL) 胆固醇通常与动脉粥样硬化发展的风险成反比1,2。虽然几项研究支持高密度脂蛋白1,2的 atheroprotective 作用, hdl 减轻动脉粥样硬化的起始和进展的机制是复杂的3,4。因此, 有人建议, HDL 的复杂结构和功能, 而不是绝对水平可以更准确地预测动脉粥样硬化5,6,7,8。一些高密度脂蛋白蛋白和脂质成分的改变会损害 hdl 的功能, 如动脉粥样硬化等炎症状态。这些 i) 降低其胆固醇流出电位9, ii) 减少消炎和增加 HDL 相关的炎蛋白6,7, iii) 降低抗氧化因子水平和活性和 HDLs抑制低密度脂蛋白氧化的能力 (LDLox)10和 iv) 增加脂质过氧化氢含量和氧化还原活性 (HDLox)9,11。对 hdl (如胆固醇外流、抗氧化功能) 的 pleotropic 功能进行评估的健壮性分析可以补充临床上 hdl-高密度脂蛋白 C 的测定。

HDL 功能通常由细胞方法评估, 如胆固醇流出试验8,12,13,14。这些方法有很大的局限性, 包括对所使用的细胞类型、报告的读数类型、缺乏标准化和甘油三酯的混杂效应7,15。这些缺点给大型临床研究带来困难16。与细胞检测相比, 无单元检测可能给 HDL 功能提供更有力的措施。胆固醇外流是 HDL 最重要的功能之一, 但它只能通过细胞检测来确定。其他方法来确定 HDL 功能, 如蛋白质组学17,18,19,20,21,22,23, 24和细胞单核蛋白的趋化性分析 HDL 功能17,22,25尚未标准化, 不能用于大规模的人类研究。

HDL 具有显著的抗氧化剂 atheroprotective 效果5,6,7,8。HDL 的抗氧化功能已确定在 LDL 的存在前细胞游离荧光分析26。这些生化荧光方法的高密度脂蛋白抗氧化功能最初开发的穆罕默德 Navab 和艾伦 Fogelman 及其同事26。虽然许多人类研究使用这些方法来确定 HDL 功能 17,18,19,20,21,22,23 ,24, 脂质 (高密度脂蛋白) 脂质 (LDL) 和脂质荧光相互作用可能会限制这些细胞游离酶生化检测 HDL 功能的重复性27,28。

最近的兴趣集中在高密度脂蛋白氧化的功能后果, 这是脂质和蛋白在 hdl 27,29,30中的氧化作用的结果。先前的研究表明, hdl 的氧化作用会损害 hdl 的功能27,29,30。高密度脂蛋白在脂质过氧化物转运中有重要作用, 高脂质过氧化物与 hdl 功能异常有关。因此高密度脂蛋白脂质过氧化物含量可用于确定 hdl 功能9,17,20,31 , 并给出已知的 hdl 函数前分析的局限性7, 15、27、32, 我们开发了一种可量化 HDL 脂质过氧化物含量 (HDLox) 32的替代荧光方法。这种方法是基于酶辣根酶 (HRP) 和荧光 Amplex 红, 可以量化 (不含胆固醇氧化酶) 的脂质过氧化物含量每毫克的 HDL-C 32。该检测的生物化学原理显示在图 1中。我们已经表明, 这种荧光方法没有先前的 HDL 函数分析的限制27,28。本试验在实验室中得到了进一步的完善和规范, 使其能够可靠地用于大规模人类研究, 即使是低温保存的等离子体32,33,34,35,36,37,38,39,40,41,42. 该方法的读数与经验证的细胞检测的读数、心血管疾病的替代措施、全身炎症、免疫功能障碍以及相关心血管和代谢风险表型有关。32,33,34,35,36,37,38,39. 在这里, 我们描述这个简单, 但稳健的方法来测量 HDL 脂质过氧化物含量 (HDLox)。这种方法可以作为一个工具, 回答重要的研究问题的作用, HDL 功能在人类疾病中的系统性炎症, 氧化应激和氧化脂有一个关键的作用 (如动脉粥样硬化)32。

Protocol

所有使用人体生物样本的实验都是在加州大学洛杉矶分校和墨尔本阿尔弗雷德医院人类伦理委员会的道德认可下进行的. 注意: 荧光 HDL 函数检测有许多变体 (请参见讨论) 32 。下面我们将描述给出最一致和可重现的结果的协议。检测的概述见 图 2 . 1. 标本处理 使用新鲜、non-hemolyzed 的血清 (可在血清分…

Representative Results

50µL 每个高密度脂蛋白样本添加到每个井和步骤7.3。50µL 的 HRP 溶液 5 u/毫升 (0.25 u), 然后添加到每个井, 在步骤7.5。样品在37° c 时孵育30分钟, 如步骤7.6 所示。50µL 的荧光试剂, 然后添加到每个井, 在步骤 7.7 (最终浓度300µM)。荧光读数 (在黑暗中), 然后评估每分钟超过120分钟, 在37° c 的荧光板阅读器 (530/590 nm 过滤器)。有代表性的荧光数据的空白, 汇集控制, 样品与已知的功?…

Discussion

这里描述的协议提供了一个强有力的工具来回答关于高密度脂蛋白功能在动脉粥样硬化和人类疾病中的作用的重要研究问题。用酶促扩法 (HRP) 对 hdl-C 的每毫克高密度脂蛋白脂质过氧化物含量进行定量分析。这种方法可以避免已知的 HDL 功能检测的局限性 (如胆固醇外流测定), 包括对所用细胞类型的显著异质性, 报告的读数类型, 缺乏标准化和混杂效应甘油三酯7,<sup class="xr…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢穆罕默德 Navab, 艾伦 Fogelman 和 Srinivasa 的工作, 他们在开发这个模型的早期迭代的关键作用。T.A.A. 得到 RMIT 大学校长博士后奖学金的支持。AJ 和 AH 得到了澳洲项目赠款1108792的支持。传统知识得到 nih 资助 nih K08AI08272、nih/NCATS 补助金µL1TR000124。

Materials

Experimental Reagents
HDL PEG (Polyethylene Glycol) Precipitating Reagent Pointe Scientific H7511
Amplex Red reagent. Life Technologies, Grand Island, NY A12216 Amplex Red Cholesterol Assay Kit.
• ≤–20°C • Desiccate
• Protect from light
DMSO. Life Technologies, Grand Island, NY A12216 Amplex Red Cholesterol Assay Kit.
• ≤–20°C • Desiccate
• Protect from light
Horse Radish Peroxidase (HRP) Life Technologies, Grand Island, NY A12216 Amplex Red Cholesterol Assay Kit.
• ≤–20°C • Desiccate
• Protect from light
Cholesterol Esterase. Life Technologies, Grand Island, NY A12216 Amplex Red Cholesterol Assay Kit.
• ≤–20°C • Desiccate
• Protect from light
 Cholesterol Reference standard Life Technologies, Grand Island, NY A12216 Amplex Red Cholesterol Assay Kit.
• ≤–20°C • Desiccate
• Protect from light
 Resorufin fluorescense Reference standard Life Technologies, Grand Island, NY A12216 Amplex Red Cholesterol Assay Kit.
• ≤–20°C • Desiccate
• Protect from light
5x Reaction Buffer. Life Technologies, Grand Island, NY A12216 Amplex Red Cholesterol Assay Kit.
• ≤–20°C • Desiccate
• Protect from light
HDL Cholesterol Automated Reagent ThermoFisher Scientific Co., San Jose, CA, USA. TR39601
Name Company Catalog Number Comments
Plasticware 
96-well plates (polypropylene, flat bottom, clear). Sigma Aldrich M0687
96-well plates (polypropylene, flat bottom, black). Sigma Aldrich M9936
1.5 mL Eppendorf tubes Eppendorf 0030 125.150
ClipTip 200, sterile ThermoFisher Scientific Co., San Jose, CA, USA. 14-488-058
Thermo Scientific Multichannel Pipettes, 8-channel, 125  ThermoFisher Scientific Co., San Jose, CA, USA.  14-387–955
Name Company Catalog Number Comments
Software 
Gen5 2.01 software Biotek, Vermont, USA NA
Name Company Catalog Number Comments
Equipment
Gen5 Plate reader Biotek, Vermont, USA NA
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Tags

Cell-freeBiochemicalFluorometricEnzymatic AssayHigh-throughputLipid PeroxidationHigh Density LipoproteinHDL FunctionCardiovascular DiseaseMetabolic AbnormalitiesInflammatory DiseasesHDL-CHDLoxPooled Quality ControlHDL Cholesterol Precipitating ReagentHRPFluorochrome ReagentDMSO
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Sen Roy, S., Nguyen, H. C. X., Angelovich, T. A., Hearps, A. C., Huynh, D., Jaworowski, A., Kelesidis, T. Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein. J. Vis. Exp. (128), e56325, doi:10.3791/56325 (2017).
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