GCx(py)GCC-IRMS for Isotope Metabolomics

用于同位素代谢组学的 GCx(py)GCC-IRMS

• 批准号: 8657070
• 负责人: JAMES T. BRENNA
• 金额: $ 30.8万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8657070
• 关键词: Acetates; Achievement; Amino Acids; Anabolism; Area; Biochemical; Biological Markers; Biomedical Research; Chemicals; Communities; Complex; Complex Mixtures; Coupled; Couples; Cytolysis; Development; Diet; 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

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.

描述（由申请人提供）：高精度同位素比质谱（IRMS）测量可以在三个基本水平上进行：批量稳定同位素分析（BSIA）、分子或化合物特异性同位素分析（CSIA）以及分子内或特定位置同位素分析（PSIA）。尽管其广泛应用于自然科学的所有领域，但还没有用于对天然同位素变异性进行高通量分子或分子内分析的仪器。最近，我们通过原理验证证明了基于 2 维 GC 的高精度综合 GCxGCC-IRMS（适用于 13C），并通过微制造与高于 1500°C 的温度兼容的高度坚固的熔融石英反应器，克服了主要的技术障碍。在这里，我们寻求利用这些突破，将 GCxGCC-IRMS 扩展到 13C 以上，以实现高通量 15N、18O 和 2H 分析，并开发高精度分子内同位素分析系统；由此产生的仪器将用于利用天然同位素和/或人工示踪剂技术研究脂肪酸和中间代谢物、乳酸和乙酸盐的假设驱动的高通量同位素代谢组学。具体目标是 (1) 设计和构建 GC-热解 xGCC-IRMS，通过采用低温调制的 GCxGCC-IRMS 进行高精度分子内分析。 (2) 2H/1H、15N/14N、18O/16O分子(CSIA)和分子内分析接口的实现；评估 2H/1H PSIA 热解后不可交换 H 的片段同位素保真度。 (3) PSIA 和 CSIA 在脂肪酸和相关分子中的应用：人类和实验系统现有样品中 n-3 多不饱和脂肪酸 (PUFA) 的天然分子和分子内同位素比率，以检验有关 n-3 和 n-3 饮食来源的假设 n-6 长链 PUFA，可能为 22:6n-3 生物合成建立一种新型生物标志物，并展示如何使用天然分子内同位素比率来测量任何膳食成分的生物转化。示踪剂。使用标记的乙酸盐、乳酸盐和 PUFA 中的 13C 和 2H 来了解脂肪酸之间 13C 通量的动力学；现有人类样本中的乳酸α13C和βD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是生物体内一个巨大的、几乎未开发的生化信息库。分子和分子内同位素是同位素代谢组学的主题，是分子前体和生理状态的天然生物标志物，其保存与代谢组学/代谢组学信息互补的信息，例如关于分子通量的信息。由于天然同位素变异性不需要事先给药或输注，因此有可能方便地应用于临床测试。最后，将快速、高灵敏度的分子内分析能力扩展到稳定同位素标记研究，扩大了常规方法在生物医学研究中的使用。