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（由适用提供）：可以在三个基本级别上进行高精度的同位素比质谱法（IRMS）测量：批量稳定同位素分析（BSIA），分子或复合特异性同位素分析（CSIA），以及分子内或位置特异性同位素分析（PSIA）。尽管在自然科学的所有领域中都有广泛的使用，但没有用于天然同位素变异性的高吞吐量分子或分子内分析的仪器。最近，我们通过以原则证明的证明为基础的二维二维基于GCXGCC-INSS并通过对高度强大的熔融熔融硅反应堆的高度兼容，我们试图通过扩大GCXGCC-ems的高度来探索这些突破？分析并通过开发用于高精度分子内同位素分析的系统；所得的仪器将用于研究假设驱动的脂肪酸和中间代谢物的高通量同位素代谢组，并具有天然同位素和/或人工示踪剂技术。具体目的是（1）通过适应低温调制的GCXGCC-IRMS设计和构建GC-Pylolysysccgcc-Irms，以进行高精度分子内分析。（2）实施2H/1H，15N/14N和18O/16O的分子（CSIA）和分子内分析；对2h/1h psia进行热解的碎片同位素保真度的评估。 N-6长链PUFA，可能建立22：6n-3生物合成的新型生物标志物，并显示如何使用天然分子内同位素比率测量任何饮食成分的生物转化。示踪剂。在标记的乙酸盐，乳酸和PUFA中使用13C和2H，以了解脂肪酸中13C通量的动力学；现有人类样品中的乳酸？13c和？d也将用于检验该分子表现出分子内同位素结构的假设。与人类健康的相关性：分子和分子内水平的天然同位素变异性是生物体中生化信息的广阔，几乎未开发的库。分子和分子内同位素（同位素代谢组学的主题）是分子前体的天然生物标志物，并且具有完整信息的物理状态的天然生物标志物（例如，有关分子通量的代谢组/替代信息）。由于天然的同位素变异性不需要事先给药或输注，因此可能在临床测试中进行方便的应用。最后，快速，高度敏感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。公共卫生相关性：为高精度分子内同位素比率（也称为位置特异性同位素分析（PSIA））在生物分子中提出了高精度的高精度和鲁棒性确定的技术，为C，N，H，H和O.提出了研究计划。对多不饱和脂肪酸和拉果酸代谢的应用是促成的。