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：大块稳定同位素分析（BSIA），分子或化合物特异性同位素分析（CSIA）和分子内或位置特异性同位素分析（PSIA）。尽管它广泛应用于自然科学的各个领域，但目前还没有用于高通量分子或分子内自然同位素变异性分析的仪器。我们最近克服了主要的技术障碍，在原理验证的基础上展示了高精度综合二维gc -based gcxgc - irms。13C，并通过微加工高度坚固的熔融硅反应器兼容温度大于1500°C。在这里，我们寻求利用这些突破，将GCxGCC-IRMS扩展到？13C实现高吞吐量？15 n, ?18O和？2H分析和开发高精度分子内同位素分析系统；由此产生的仪器将应用于研究假说驱动的脂肪酸和中间代谢物，乳酸和醋酸盐的高通量同位素代谢组学，使用天然同位素和/或人工示踪技术。具体目的是：(1)采用低温调制的gcxgc - irms，设计并构建用于分子内高精度分析的gc -热解xgc - irms。(2)实现分子（CSIA）和分子内2H/1H、15N/14N、18O/16O分析接口；2H/1H非交换h PSIA裂解后片段同位素保真度评价(3)PSIA和CSIA在脂肪酸及相关分子中的应用：在现有的人体和实验系统样品中，研究n-3多不饱和脂肪酸（PUFA）的天然分子和分子内同位素比率，以验证关于n-3和n-6长链PUFA的膳食来源的假设，可能建立22:6n-3生物合成的新生物标志物，并展示如何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13C和？在标记的乙酸、乳酸和PUFA中添加2H，以了解脂肪酸之间13C通量的动力学；乳酸？13C和？现有人类样本中的D也将用于测试该分子具有分子内同位素结构的假设。与人类健康的相关性：在分子和分子内水平上的自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，具有与代谢组学/代谢组学信息互补的信息，例如分子通量。由于自然同位素变化不需要预先给药或输注，因此很可能方便地应用于临床试验。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，扩展了常规方法在生物医学研究中的应用。