Slow-MAS NMR Metabolomics

描述(申请人提供)：高分辨率魔角旋转(hr-MAS)核磁共振，使用几千赫或更高的样品自转速度，已成为完整生物组织代谢图谱的强大工具。然而，有几个关键问题需要解决，以使MAS核磁共振广泛应用于生物医学、临床和翻译研究。首先，HR-MAS技术是破坏性的，因为与快速样品旋转相关的大离心力。其次，由于与快速纺纱相关的各种技术挑战，HR-MAS实验中的样品量也被限制在~15到60毫升。我们的研究目标是开发一种非破坏性、高分辨率和高灵敏度的MAS-核磁共振方法，作为对代谢组学研究的补充。为了达到我们的目标，我们制定了两个具体目标。目的1：建立一种非破坏性的慢样品旋转MAS核磁共振代谢组学技术，包括500 MHz核磁共振波谱仪上的Slow-MAS探针和转子位置同步的Slow-MAS脉冲序列。Slow-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，使用单一探针，体积从小到200纳升(NL)到大到1000微升(1.0厘米3)或更大。NL的能力将使其有可能通过对单个小型实验动物的持续研究，并最终在患者身上，使用微创组织活检和血液样本，在很长一段时间内跟踪代谢变化。微升到立方厘米的能力将满足在不同大小的完整生物组织上进行代谢分析的广泛需求，从而使在完整组织上进行大规模代谢分析成为可能。我们已经成功地在300 MHz核磁共振光谱仪上使用概念验证的Slow-MAS核磁共振探针进行了概念验证的实验，以证明我们提出的研究是正确的。目的2：NL Slow-MAS方法的应用。我们将应用Slow-MAS探针的NL特征，通过对8只肥胖C57BL/6小鼠和8只正常C57BL/6小鼠(对照组)进行体积为200至500 NL的微创骨骼肌和血液样本的微创活检，持续跟踪代谢变化，以确定与肥胖相关的可能代谢物生物标志物。在In Life抽样结束时， 小鼠将被处死，整个器官将使用探测器的厘米3特征进行研究。我们还将对从肥胖加速的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。肥胖已经成为一种公认的代谢紊乱的危险因素，尤其是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紊乱途径还没有得到很好的证实。这主要是由于对非常少量的组织样本进行直接代谢物分析的困难。我们的NL Slow-MAS能力将使从饮食诱导的肥胖+动脉粥样硬化小鼠模型中切除的完整动脉上的代谢图谱成为可能。

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.