Development of a Novel Technology for Preparative Fractionation and Characterization of Lipoprotein Particles

项目摘要 高密度脂蛋白(高密度脂蛋白)是唯一最强的长寿预测因子，可预防多种疾病 疾病，从慢性疾病，如心血管疾病和神经退化，到急性感染和 败血症死亡率，以及介于两者之间的一切。如果我们能正确地摄取高密度脂蛋白，我们就能健康长寿。然而，尽管 经过50多年的研究，高密度脂蛋白一直是提高高密度脂蛋白的谜团和治疗方法。 功能被证明是难以捉摸的。高密度脂蛋白高度异质性，很难分离和表征，因为 它们的胶体性质，多分子性质，但尺寸很小(直径20 nm)。取得进展的一个关键障碍是 缺乏同时量化高密度脂蛋白颗粒的大小和数量并将它们分离出来的技术 它们保持完好，可以进行各种成分和功能分析。目标 该项目的目的是获得有关使用尺寸排除的仪器应用的新技术知识 与多个在线静态和动态光学检测器相结合的层析，并测量 这项技术相对于最先进的分离和物理化学方法的定量优势 高密度脂蛋白颗粒的表征。特别是，这个项目将解决量化数字的关键问题 使用非破坏性方法同时测量颗粒并通过以下方式对颗粒进行分级 大小，从而可以计算基于大小的不同HDL子类在 每粒子基础。目前，研究人员受到一个简单问题的限制，即没有一个好的分母： 唯一的选择是在高密度脂蛋白中表达一个重要成分或功能容量的量 根据“浓度”的粗略替代物(例如，分离部分中的总蛋白)进行测量。因此， 如果样本A和B中有更多的某种蛋白质(或更高的功能能力)，就没有办法区分 这是否仅仅是因为样品A中含有更多的粒子，或者是否存在更多的分子 每个颗粒的蛋白质(或更高的功能容量)。不同大小的高密度脂蛋白携带不同的绝对和相对 单个蛋白质、脂类和其他成分的数量，从少至两个分子的主要 载脂蛋白、载脂蛋白A-I和12个分子的胆固醇酯中最小的高密度脂蛋白，多达4-6个 载脂蛋白A-I分子和数百个胆固醇酯分子，在 其他具有数十种功能的关键成分的浓度，从抗氧化剂到 免疫调节、抗蛋白水解剂等等。而且因为颗粒大小决定了与 受体，清除率，甚至可能高密度脂蛋白是否能通过血脑屏障，知道这个数字 不同大小的颗粒，以及它们所载货物的每种颗粒组成对 开发敏感、可操作的诊断方法和有针对性的、有效的治疗方法。因此，这项技术 在这个项目中开发的将深刻地使生物医学研究团体能够回答关键问题 关于高密度脂蛋白功能生物学，涉及广泛的临床和治疗应用。

Project Summary High-density lipoproteins (HDL) are the single strongest predictors of longevity and protect against a wide array of diseases, from chronic conditions like cardiovascular disease and neurodegeneration, to acute infection and sepsis mortality, and everything in between. If we can get HDL right, we can live long, healthy lives. Yet despite over 50 years of research, HDL have remained an enigma and therapeutic approaches for improving HDL function have proven elusive. HDL are highly heterogeneous and difficult to isolate and characterize because of their colloidal, multi-molecular nature yet very small size (< 20 nm in diameter). A critical barrier to progress is the lack of technologies to simultaneously quantify the size and number of HDL particles, and isolate them such that they remain intact and amenable for a variety of both compositional and functional analyses. The objectives of this project are to gain new technological knowledge on the application of our instrument using size exclusion chromatography coupled with multiple inline static and dynamic optical detectors, and to measure the quantitative advantages of this technology over state-of-the-art approaches for the isolation and physicochemical characterization of HDL particles. In particular, this project will solve the critical problem of quantifying the number of particles using a non-destructive approach that simultaneously measures and fractionates the particles by size, making it possible to evaluate the function and composition of different size-based HDL subclasses on a per particle basis. Currently, researchers are limited by the simple problem of not having a good denominator: the only option is to express the amount of an important constituent or functional capacity in the HDL we have measured based on a rough substitute for “concentration” (e.g. total protein in the isolated fraction). Therefore, if there is more of a certain protein (or higher functional capacity) in sample A vs. B, there is no way to distinguish whether that is simply because sample A has more particles in it or whether there are more molecules of that protein (or higher functional capacity) per particle. Different sizes of HDL carry different absolute and relative amounts of individual proteins, lipids, and other components, from as few as 2 molecules of the main apolipoprotein, apolipoprotein A-I, and 12 molecules of cholesteryl ester in the smallest HDL, to as many as 4-6 molecules of apolipoprotein A-I and hundreds of molecules of cholesteryl ester, with similar variability in the concentrations of other critical components that confer dozens of functions, from antioxidant, to immunomodulatory, to anti-proteolytic to name a few. And because particle size determines binding affinity to receptors, clearance rates, and likely even whether HDL can cross the blood-brain-barrier, knowing the number of particles of different sizes, and also the per particle composition of the cargo they carry is critical to the development of sensitive, actionable diagnostics, and targeted, effective therapeutics. Thus, the technology developed in this project will profoundly enable the biomedical research community to answer critical questions about HDL functional biology across a broad array of clinical and therapeutic applications.