GCx(py)GCC-IRMS for Isotope Metabolomics

用于同位素代谢组学的 GCx(py)GCC-IRMS

• 批准号: 7945900
• 负责人: JAMES T. BRENNA
• 金额: $ 35.26万
• 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 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7945900
• 关键词: Acetates; Achievement; Amino Acids; Anabolism; Area; Biochemical; Biological Markers; Biomedical Research; Chemicals; Communities; Complex Mixtures; Coupled; Couples; Cytolysis; Development; Dietary Component; Dose; Evaluation; Exhibits; Face; Fatty Acids; Gas Chromatography; Gases; Goals; Health; Human; Individual; Infusion procedures; Isotopes; Kinetics; Knowledge; Label; Laboratories; Lactic acid; Manufacturer Name; Mass Spectrum Analysis; Measurement; Measures; Metabolic Biotransformation; Metabolism; Methods; Molecular; Molecular Analysis; 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. 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（由申请人提供）：可以在三个基本水平上进行高精度同位素比质谱（IRMS）测量：批量稳定同位素分析（BSIA）、分子或化合物特定同位素分析（CSIA）和分子内或位置特定同位素分析（PSIA）。尽管其在自然科学的所有领域中广泛使用，但没有用于天然同位素变异性的高通量分子或分子内分析的仪器。我们最近克服了主要的技术障碍，证明了高精度的全面的二维GC为基础的GCxGCC-IRMS的原理的基础上？13 ℃，并通过微制造高度坚固的熔融石英反应器，与高于1500 ℃的温度兼容。在这里，我们寻求利用这些突破，扩大GCxGCC-IRMS超越？13 C实现高吞吐量？15 N，？18、？2 H分析，并通过开发一个高精度的分子内同位素分析系统，得到的仪器将被应用于研究假设驱动，高通量的脂肪酸和中间代谢产物，乳酸盐和乙酸盐的同位素代谢组学，与天然同位素和/或人工示踪技术。具体目的是（1）通过对低温调节的GCxGCC-IRMS的调整，设计和构建用于高精度分子内分析的GC-pyrolysisxGC-IRMS。(2)实现2 H/1H、15 N/14 N和18 O/16 O的分子（CSIA）和分子内分析接口;评估热解后2 H/1H PSIA对非交换性H的碎片同位素保真度。(3)PSIA和CSIA在脂肪酸和相关分子中的应用：来自人类和实验系统的现有样本中n-3多不饱和脂肪酸（PUFA）的天然分子和分子内同位素比率，以测试关于n-3和n-6长链PUFA饮食来源的假设，可能建立22：6 n-3生物合成的新生物标志物，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。追踪器使用13 C和？2 H标记的乙酸盐，乳酸盐和PUFA，以了解脂肪酸之间的13 C通量的动力学;乳酸？13 C和？在现有的人类样本中的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天然同位素变异性是生物体内几乎未开发的巨大生化信息库。分子和分子内同位素，同位素代谢组学的主题，是分子前体和生理状态的天然生物标志物，其保持与代谢组学/代谢组学信息互补的信息，例如，关于分子通量。由于天然同位素的变异性不需要预先给药或输注，因此很可能方便地应用于临床检测。最后，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应用。 公共卫生关系：针对C、N、H和O提出了生物分子中高通量和高精度分子内同位素比稳健测定的技术，也称为位置特异性同位素分析（PSIA）。该研究计划利用全面的二维气相色谱仪和创新组件的适应性来创建一个强大的系统。提出了在多不饱和脂肪酸和乳酸代谢中的应用.