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Development of a nanoliter slow-MAS NMR metabolomics probe

纳升慢 MAS NMR 代谢组学探针的开发

基本信息

批准号：
7896628
负责人：
Jian Zhi Hu
金额：
$ 33.53万
依托单位：
BATTELLE PACIFIC NORTHWEST LABORATORIES
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2012-07-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7896628
关键词：
Age Animals Biochemistry Biological Biological Markers Biometry Biopsy Biopsy Specimen Blood Blood capillaries Blood specimen Body Fluids C57BL/6 Mouse Cells Data Development Diagnosis Diet Disease Evolution Frequencies Gender Gene Expression Goals Hand Health Individual Investigation Laboratory Animals Magic Magnetic Resonance Imaging Magnetism Metabolic Metabolic Diseases Methods Mus Muscle NMR Spectroscopy Nuclear Magnetic Resonance Obese Mice Obesity Organ Outcome Pathway interactions Patients Performance Physiologic pulse Predisposition Preparation Public Health Radio Research Resolution Risk Factors Sample Size Sampling Skeletal Muscle Staging System Systems Biology Techniques Time Tissue Sample Tissues Tube Urine Variant analytical tool biological systems capillary cell type high standard magnetic field metabolomics milliliter minimally invasive mouse model nanolitre novel public health relevance success time use tool

项目摘要

DESCRIPTION (provided by applicant): Metabolomics is a powerful new systems biology tool that is capable of simultaneously investigating multiple biological pathways, detecting and diagnosing a disease and evaluating the efficacy of a therapy at an early stage. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the leading metabolomics tools. Tissue metabolic profiling is of significance because a disease is often associated with a localized tissue/organ malfunction. The technique of high resolution magic angle spinning (hr-MAS) has been increasingly used for metabolic profiling of intact tissues with notable successes. However, the requirement of tissue mass of 10- 30mg or more for standard hr-MAS NMR metabolomics analysis may present a problem in studying small laboratory animals such as mice, where animals need to be sacrificed to obtain adequate amount of tissue for subsequent analysis. Furthermore, a tissue mass of 10-30mg could encompass many different cell types and study on a smaller sample size would be desired in terms of cell biochemistry. The goal of our research is to develop a novel high resolution, high sensitivity method for NMR metabolomics investigations of tissue samples with mass/volume as small as 300<g/300nL. The outcome of the project will make it possible to carry out a continued investigation on a single small laboratory animal over time using minimally invasive tissue biopsy samples. To reach our goal, we have formulated two specific Aims. Aim 1: Development of a slow magic angle sample spinning NMR probe with sample volume as small as 300 nanoliters (nL). We will use a combination of micro-RF-coil with magnetic susceptibility matched wires, and capillary sample tube to obtain the optimal sensitivity. The spectral resolution will be further enhanced by integrating our established slow-MAS 1H NMR method applied at a sample spinning rate of about 80Hz. With slow-MAS technique, the line broadening due to magnetic susceptibility gradients within tissue cells, between the sample and the sample tube are eliminated, giving rise to a high resolution 1H NMR spectrum. We will expand the applicability of the probe such that tissue samples from 300nL to several mL can also be investigated. Initially, a probe will be built to operate at 7.05T field, followed by a second probe that will operate at 11.7 T field so the potential advantages of slow-MAS at higher magnetic field strengths can be explored. Aim 2: Application of the nL slow-MAS probe. We will carry out a comprehensive NMR metabolomics investigation using minimally invasive biopsy muscle samples of nL in volume (including the use of blood and non-invasive urine samples of <l or less as a supplementary method to standard NMR metabolomics) obtained from normal vs obese C57BL/6 mice from about 6 weeks to about 16 weeks of age to identify possible metabolite biomarkers that are related to obesity. The time variation of metabolite biomarkers for each animal will be determined. The results will be compared between the animals within the same group, from which the normal biological variations will be determined and the advantages of following a single animal over time period will be demonstrated. PUBLIC HEALTH RELEVANCE: We propose to develop a novel slow magic angle sample spinning probe that is capable of high resolution and high sensitivity metabolic profiling on biological samples, in particular tissue samples, with volume as small as 300 nanoliters (nL) to sample volume as large as a few milliliters. 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.

描述（由申请人提供）：代谢组学是一种强大的新型系统生物学工具，能够同时研究多种生物学途径，检测和诊断疾病，并在早期阶段评估治疗效果。核磁共振（NMR）波谱是主要的代谢组学工具之一。组织代谢谱分析具有重要意义，因为疾病通常与局部组织/器官功能障碍有关。高分辨率魔角旋转（hr-MAS）技术已越来越多地用于完整组织的代谢谱分析，并取得了显著的成功。然而，标准的hr-MAS NMR代谢组学分析需要10- 30mg或更多的组织质量，这在研究小型实验动物（如小鼠）时可能会出现问题，在这些实验动物中，需要牺牲动物以获得足够数量的组织用于后续分析。此外，10-30mg的组织质量可以包含许多不同的细胞类型，并且在细胞生物化学方面需要更小的样本量进行研究。我们的研究目标是开发一种新的高分辨率、高灵敏度的方法，用于质量/体积小至300<g/300nL的组织样品的核磁共振代谢组学研究。该项目的结果将使对单个小型实验动物进行持续研究成为可能，并使用微创组织活检样本进行长期研究。为实现这一目标，我们制定了两个具体目标。目标1：开发一种样品体积小至300纳升的慢魔角样品旋转核磁共振探针。我们将使用微射频线圈与磁化率匹配的导线和毛细管样管的组合来获得最佳灵敏度。通过整合我们建立的慢mas 1H NMR方法，在约80Hz的样品旋转速率下，光谱分辨率将进一步提高。使用慢- mas技术，消除了组织细胞内、样品和样管之间由于磁化率梯度引起的谱线展宽，从而产生高分辨率的1H NMR谱。我们将扩大探针的适用性，使组织样本从300nL到几mL也可以调查。首先，将建造一个在7.05T磁场下工作的探测器，然后再建造一个在11.7 T磁场下工作的探测器，以便在更高磁场强度下探索慢速mas的潜在优势。目的2:nL慢- mas探针的应用。我们将进行全面的核磁共振代谢组学研究，使用从6周龄至16周龄的正常和肥胖C57BL/6小鼠中获得的nL体积的微创活检肌肉样本（包括使用<l或更少的血液和非侵入性尿液样本作为标准核磁共振代谢组学的补充方法），以确定可能与肥胖相关的代谢物生物标志物。将确定每种动物的代谢物生物标志物的时间变化。结果将在同一组动物之间进行比较，从中确定正常的生物学变化，并证明长期跟踪单个动物的优势。