Slow-MAS NMR Metabolomics

慢速 MAS NMR 代谢组学

基本信息

批准号：
8416150
负责人：
Jian Zhi Hu
金额：
$ 41.1万
依托单位：
BATTELLE PACIFIC NORTHWEST LABORATORIES
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-15 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8416150
关键词：
Address Age Arteries Atherosclerosis Biochemical Biological Biological Markers Biomedical Research Biopsy Biopsy Specimen Blood specimen C57BL/6 Mouse Clinical Clinical Research Complement Data Detection Development Diet Disease Extravasation Gene Expression Goals Insecta Investigation Knowledge Laboratories Laboratory Animals Learning Life Liquid substance Magic Metabolic Metabolic Diseases Metabolic Pathway Methods Mus National Institute of Environmental Health Sciences Obese Mice Obesity Operative Surgical Procedures Organ Outcome Patients Performance Physiologic pulse Positioning Attribute Proteomics Protocols documentation Research Resolution Risk Factors Sampling Skeletal Muscle Techniques Technology Time Tissue Sample Tissues Translational Research Work base cell type design improved magnetic field metabolomics milliliter minimally invasive mouse model nanolitre novel research clinical testing research study seal success time use tool

项目摘要

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. PUBLIC HEALTH RELEVANCE: We propose to develop a non-destructive magic angle spinning metabolomics technique that is capable of high resolution and high sensitivity metabolic profiling on biological samples, in particular, on tissue samples with sample volume from as small as 200 nanoliters (nL) to as large as a milliliter or more using a single probe and using only a few minutes. If successful, this technique will enable large scale metabolic profiling on intact biological tissues of various sizes that will have wide application in biomedical, clinicl and translational researches.

描述（由申请人提供）：使用几kHz或更高的样品旋转速率的高分辨率魔角旋转（hr-MAS）NMR已成为完整生物组织代谢分析的有力工具。然而，为了使 MAS NMR 广泛应用于生物医学、临床和转化研究，有一些关键问题需要解决。首先，hr-MAS 技术由于与快速样品旋转相关的大离心力而具有破坏性。其次，由于与快速旋转相关的各种技术挑战，hr-MAS 实验中的样品体积被限制在约 15 至 60 ml。我们研究的目标是开发一种无损、高分辨率和高灵敏度的 MAS-NMR 方法，作为代谢组学研究中 hr-MAS 的补充。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开发非破坏性 MAS NMR 代谢组学技术，包括 500 MHz NMR 波谱仪上的慢速 MAS 探针和转子位置同步慢速 MAS 脉冲序列。 Slow-MAS 技术将能够使用单个探针对体积从小至 200 纳升 (nL) 到大至 1000 微升 (1.0 cm3) 或以上的生物组织样本进行高分辨率和高灵敏度的代谢分析。 nL 能力将使通过对单个小型实验动物进行持续研究，并最终在很长一段时间内使用微创组织活检和血液样本来跟踪代谢变化成为可能。微升到立方厘米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。我们使用经过概念验证的慢速 MAS NMR 探头，在 300 MHz NMR 波谱仪上成功进行了经过概念验证的实验，以证明我们提出的研究的合理性。目标 2：nL Slow-MAS 方法的应用。我们将应用慢速 MAS 探针的 nL 特征，使用微创活检骨骼肌和体积为 200 至 500 nL 的血液样本，连续跟踪 8 只肥胖 C57BL/6 小鼠和 8 只正常 C57BL/6 小鼠的代谢变化（对照）超过 8 至 16 周，以确定与肥胖相关的可能代谢生物标志物。在生命采样结束时，将处死小鼠并使用探针的 cm3 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尚不明确。这主要是由于对极少量的组织样本进行直接代谢分析的困难。我们的 nL Slow-MAS 能力将使对从饮食诱导的肥胖 + 动脉粥样硬化小鼠模型中切除的完整动脉进行代谢分析成为可能。公共健康相关性：我们建议开发一种非破坏性魔角旋转代谢组学技术，能够对生物样品，特别是样品量小至 200 纳升 (nL) 的组织样品进行高分辨率和高灵敏度的代谢分析。使用单个探针仅需几分钟即可测量到毫升或更多。如果成功，该技术将实现大规模代谢分析各种尺寸的完整生物组织，将在生物医学、临床和转化研究中广泛应用。