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GCx(py)GCC-IRMS for Isotope Metabolomics

用于同位素代谢组学的 GCx(py)GCC-IRMS

基本信息

批准号：
8109892
负责人：
JAMES T. BRENNA
金额：
$ 34.91万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8109892
关键词：
Acetates Achievement Amino Acids Anabolism Area Biochemical Biological Markers Biomedical Research Chemicals Communities Complex Complex Mixtures Coupled Couples Cytolysis Development Dietary Component Dose Evaluation Exhibits Face Fatty Acids Gas Chromatography Goals Health Human Individual Infusion procedures Isotopes Kinetics Knowledge Label Laboratories Lactic acid Manufacturer Name Mass Fragmentography Mass Spectrum Analysis Measurement Measures Metabolic Biotransformation Metabolism Methods Molecular Molecular Analysis Molecular Biology Natural Sciences Nutritional Organism Physiological Plant Leaves Plants Polyunsaturated Fatty Acids Positioning Attribute Research Resolution Sampling Silicon Dioxide Stable Isotope Labeling Structure System Techniques Technology Temperature Testing Tracer Work base branched chain fatty acid design and construction high throughput technology innovation instrument instrumentation interest metabolomics novel nutrition public health relevance repository research clinical testing stable isotope tool two-dimensional

项目摘要

DESCRIPTION (provided by applicant): High precision isotope ratio mass spectrometry (IRMS) measurements can be made on three fundamental levels: bulk stable isotope analysis (BSIA), molecular or compound-specific isotope analysis (CSIA), and intramolecular or position-specific isotope analysis (PSIA). Despite its widespread use in all areas of natural science, there are no instruments for high throughput molecular or intramolecular analysis of natural isotope variability. We have recently overcome major technical barriers by demonstrating on a proof-of-principle basis high precision comprehensive 2-dimensional-GC-based GCxGCC-IRMS for ?13C and by microfabricating highly robust fused silica reactors compatible with temperatures greater than 1500¿C. Here, we seek to exploit these breakthroughs by expanding GCxGCC-IRMS beyond ?13C to enable high throughput ?15N, ?18O and ?2H analyses and by developing a system for high precision intramolecular isotope analysis; the resulting instrumentation will be applied to investigate hypothesis-driven, high throughput isotopic metabolomics of fatty acids and intermediary metabolites, lactate and acetate, with natural isotope and/or artificial tracer techniques. The Specific Aims are (1) Design and construction of a GC-pyrolysisxGCC-IRMS for high precision intramolecular analysis by adaptation of a cryogenically modulated GCxGCC-IRMS. (2) Implementation of interfaces for molecular (CSIA) and intramolecular analysis of 2H/1H, 15N/14N, and 18O/16O; Evaluation of fragment isotopic fidelity after pyrolysis for 2H/1H PSIA for non-exchangeable H. (3) Applications of PSIA and CSIA to fatty acids and related molecules: Natural molecular and intramolecular isotope ratios of n-3 polyunsaturated fatty acids (PUFA) in existing samples from humans and experimental systems to test hypotheses regarding dietary origin of n-3 and n-6 long chain PUFA, possibly establishing a novel biomarker for 22:6n-3 biosynthesis, and showing how biotransformation of any dietary component can be measured with natural intramolecular isotope ratios. Tracers. Use of 13C and ?2H in labeled acetate, lactate, and PUFA to understand the kinetics of 13C flux among fatty acids; lactic acid ?13C and ?D in existing human samples will also be used to test the hypothesis that this molecule exhibits intramolecular isotopic structure. Relevance to human health: Natural isotope variability at the molecular and intramolecular levels is a vast, nearly untapped repository of biochemical information within organisms. Molecular and intramolecular isotopes, the subject of isotopic metabolomics, are natural biomarkers of molecular precursors, and of physiological state that hold information complementary to metabolomic/metabonomic information, for instance, about molecular flux. Because natural isotopic variability requires no prior dosing or infusions, convenient applications to clinical testing are likely. Finally, the expansion of rapid, highly sensitive intramolecular analysis capabilities to stable isotope labeling studies expands the use of a routine method in biomedical research. PUBLIC HEALTH RELEVANCE: Enabling technology for high throughput and robust determination of high precision intramolecular isotope ratios, also known as position-specific isotope analysis (PSIA), in biomolecules is proposed for C, N, H, and O. The research plan capitalizes on adaptation of comprehensive two dimensional gas chromatography instrumentation as well as innovative components to create a robust system. Applications to polyunsaturated fatty acid and lactic acid metabolism are pro- posed.

描述(申请人提供)：高精度同位素比质谱仪(IRMS)可在三个基本层面上进行测量：整体稳定同位素分析(BSIA)、分子或化合物特定同位素分析(CSIA)以及分子内或特定位置同位素分析(PSIA)。尽管它在自然科学的所有领域都得到了广泛的应用，但还没有高通量的分子或分子内分析天然同位素变异性的仪器。我们最近克服了主要的技术障碍，通过在原理验证的基础上展示了高精度的、全面的、基于2维GC的GCxGCC-IRMS，并通过微制造兼容高于1500℃的温度的高度坚固的熔融二氧化硅反应器。在这里，我们寻求利用这些突破，通过将GCxGCC-IRMS扩展到？13℃以实现高通量？15N、？18O和？2H分析，并通过开发高精度分子内同位素分析系统；所产生的仪器将用于研究假设驱动的、高通量的脂肪酸和中间代谢物、乳酸盐和醋酸盐的同位素代谢组学，并使用天然同位素和/或人工示踪技术。具体目标是：(1)利用低温调制的GC-GCC-IRMS，设计和构建用于高精度分子内分析的GC-热裂解×GCC-IRMS。(2)实现了2H/1H、15N/14N和18O/16O的分子(CSIA)和分子内分析接口；对非交换性H的2H/1H PSIA裂解后片段同位素保真度的评估(3)PSIA和CSIA在脂肪酸及相关分子上的应用：现有人类样本和实验系统中n-3多不饱和脂肪酸(PUFA)的自然分子和分子内同位素比率，以测试有关n-3和n-6长链PUFA饮食来源的假说，可能建立一种新的22：6n-3生物合成生物标志物，并展示如何用天然分子内同位素比率来衡量任何膳食成分的生物转化。追踪器。利用标记的醋酸盐、乳酸盐和多不饱和脂肪酸中的~(13)C和~2H来了解脂肪酸之间的~(13)C和~(13)D的流动动力学，也将利用已有的人体样品中的~(13)C和~(13)D来检验该分子具有分子内同位素结构的假设。与人类健康相关：分子和分子内水平上的自然同位素可变性是生物体内一个巨大的、几乎未被开发的生化信息储存库。作为同位素代谢组学的主题，分子和分子内同位素是分子前体和生理状态的天然生物标志物，持有与代谢组学/代谢组学信息互补的信息，例如关于分子通量的信息。由于天然同位素的可变性不需要事先给药或输液，因此可以方便地应用于临床测试。最后，将快速、高灵敏的分子内分析能力扩展到稳定的同位素标记研究，扩大了生物医学研究中常规方法的使用。与公共健康相关：针对C、N、H和O提出了高通量和稳健地测定生物分子中高精度分子内同位素比率的技术，也称为位置特异性同位素分析(PSIA)。研究计划利用全面的二维气相色谱仪器的适应性以及创新的组件来创建稳健的系统。提出了其在多不饱和脂肪酸和乳酸代谢中的应用。