Slow-MAS NMR Metabolomics

描述（由申请人提供）：高分辨率魔角旋转（hr-MAS）NMR，其中使用几kHz或更高的样品旋转速率，已成为完整生物组织代谢谱分析的有力工具。然而，有一些关键问题需要解决，以MAS NMR被广泛用于生物医学，临床和转化研究。首先，hr-MAS技术是破坏性的，由于与快速样品旋转相关的大离心力。其次，hr-MAS实验中的样品体积被限制在约15至60 ml，这也是由于与快速旋转相关的各种技术挑战。我们的研究目标是开发一种非破坏性的，高分辨率和高灵敏度的MAS-NMR方法，补充hr-MAS的代谢组学研究。为了实现我们的目标，我们制定了两个具体目标。目标1：通过使用慢速样品旋转，包括在500 MHz NMR光谱仪上的慢速MAS探针和转子位置同步的慢速MAS脉冲序列，开发非破坏性MAS NMR代谢组学技术。慢MAS技术将能够对生物组织样本进行高分辨率和高灵敏度的代谢分析，其体积从小至200纳升（nL）到大至1000微升（1.0 cm 3）或更多。nL能力将使人们有可能通过对单个小型实验室动物的持续研究，并最终对患者进行长期的微创组织活检和血液样本，来跟踪代谢变化。微升至cm 3的能力将满足对不同大小的完整生物组织进行代谢分析的广泛需求，从而能够对完整组织进行大规模代谢分析。我们已经成功地进行了概念验证实验的300兆赫核磁共振光谱仪使用的概念验证慢MAS核磁共振探针，以证明我们提出的研究。目的2：nL慢MAS方法的应用。我们将应用慢MAS探针的nL特征，使用8只肥胖C57 BL/6小鼠和8只正常C57 BL/6小鼠（对照）的200至500 nL体积的微创活检骨骼肌和血液样本，在8至16周龄期间连续跟踪代谢变化，以鉴定与肥胖相关的可能的代谢物生物标志物。在活体取样结束时， 将处死小鼠并使用探针的cm 3特征研究整个器官。我们还将对从肥胖加速动脉粥样硬化小鼠模型中切除的动脉进行慢MAS代谢组学研究。肥胖已成为多种代谢紊乱的公认危险因素，特别是包括动脉粥样硬化性心血管疾病。然而，导致肥胖加速动脉粥样硬化的代谢途径紊乱还没有很好地建立。这主要是由于在非常少量的组织样品上进行直接代谢谱分析的困难。我们的nL慢- MAS能力将使从饮食诱导的肥胖+动脉粥样硬化小鼠模型切除的完整动脉上的代谢谱分析成为可能。

DESCRIPTION (provided by applicant): High resolution magic angle spinning (hr-MAS) NMR where a sample spinning rate of a few kHz or more is used has become a powerful tool for metabolic profiling of intact biological tissues. However, there are a few critical issues that nee be addressed in order for MAS NMR to be used widely in biomedical, clinical, and translational researches. First, hr-MAS technique is destructive due to the large centrifugal force associated with fast sample spinning. Second, the sample volume in an hr-MAS experiment is restricted to ~15 to 60 ml also due to a variety of technical challenges associated with fast spinning. The goal of our research is to develop a non-destructive, high resolution and high sensitivity MAS-NMR method that complements hr-MAS for metabolomics investigations. To reach our goal, we have formulated two specific Aims. Aim 1: Development of a non-destructive MAS NMR metabolomics technique by using slow sample spinning, including a slow-MAS probe and rotor position synchronized slow-MAS pulse sequences on a 500 MHz NMR spectrometer. The slow-MAS technology will be capable of high resolution and high sensitivity metabolic profiling on biological tissue samples with volume variable from as small as 200 nanoliters (nL) to as large as 1000 microliters (1.0 cm3) or more using a single probe. The nL capability will make it possible to follow the metabolic changes through a continued investigation on a single small laboratory animal, and ultimately on a patient, over a long period of time using minimally invasive tissue biopsy and blood samples. The micro-liter to cm3 capability will serve the wide spread need of metabolic profiling on intact biological tissues of variable sizes, thus enabling large scale metabolic profiling on intact tissues. We have successfully performed concept- proven experiment on a 300 MHz NMR spectrometer using a concept-proven slow-MAS NMR probe to justify our proposed research. Aim 2: Application of the nL slow-MAS method. We will apply the nL feature of the slow-MAS probe to continuously follow the metabolic changes using minimal invasive biopsy skeletal muscle and blood samples of 200 to 500 nL in volume on 8 obese C57BL/6 mice, and 8 normal C57BL/6 mice (controls) over ages 8 to 16 weeks to identify possible metabolite biomarkers that are related to obesity. At the end of the in life sampling, the mice will be sacrificed and whole organs will be studied using the cm3 feature of the probe. We will also carry out slow-MAS metabolomics studies on artery excised from an obese- accelerated atherosclerosis mouse model. Obesity has become a recognized risk factor for a variety of metabolic disorders, including in particular atherosclerotic cardiovascular diseases. However, the disordered metabolic pathways that contribute to obesity-accelerated atherosclerosis are not well established. This is mainly due to the difficulties of direct metaboli profiling on very small amount of tissue samples. Our nL slow- MAS capability will make the metabolic profiling on the intact artery excised from a diet-induced obese + atherosclerosis mouse model possible.