COMPREHENSIVE ANALYSIS OF METABOLIC FLUXES IN VIVO

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 尽管分子生物学的最新进展使人们对疾病的转录基础有了更全面的了解，但允许在体内测量转录最终产物（代谢流）的技术非常有限。动物模型中酶表达的变化与通过酶的通量的变化不匹配的许多例子强调了测量通量的重要性。因此，如果要明确了解疾病的代谢基础，则必须进一步开发测量体内通量的技术，以便基础和临床科学家可以轻松地利用它们。小分子代谢物的 NMR 同位素异构体分析非常适合进行这些测量，因为化学位移和自旋-自旋耦合可以很好地定义同位素异构体群体。了解代谢物中的同位素分布就等于了解产生同位素异构体的生化途径的通量。核心 I 的持续重点是解决基于 NMR 的代谢分析的技术开发，重点是提高体内代谢的 NMR 同位素异构体分析的灵敏度。具体目标是： 1. 多重代谢通量。该目标旨在通过将肝通量的额外 13 C 示踪剂纳入上一个资助周期开发的技术中，扩大在单个体内实验中测量的代谢途径的数量。这一目标将通过同时测量多个肝通量来提高 NMR 同位素异构体方法的实验效率（与其他同位素检测方法相比）。 2. 复杂系统示踪数据的数学分析。我们的目标是更全面地完善描述肝通量的参数敏感性、实验设计的数学模型，并将现有的动力学模型从简单的单室组织模型扩展到更现实的两室模型。随着我们在新型高场人体扫描仪上开展更多体内工作，这将增强我们使用 13C NMR 多重数据分析肝脏和大脑代谢的能力。 3. 改进代谢研究中 2H 和 13C 的 NMR 检测。为了提高代谢通量测量的适用性，我们必须继续提高 2H 和 13C NMR 实验的灵敏度，这是该方法的支柱。我们将 2H NMR 和 JHSQC 实验扩展到 18.8T，以利用更高磁场的更高灵敏度和色散，并开发必要的方法，以最大限度地利用新型微螺线管探针，为样本有限的情况提供更高的灵敏度。最后，我们将测试新型高温超导核磁共振探针用于代谢测量的适用性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite recent advances in molecular biology that have provided a fuller understanding of the transcriptional basis of disease, technology that allows the ultimate end product of transcription, metabolic flux, to be measured in vivo is very limited. The importance of measuring flux is highlighted by a number of examples in animal models where changes in enzyme expression do not match changes in flux through the enzyme. Thus, if the metabolic basis of disease is to be explicitly understood, techniques which measure in vivo fluxes must be further developed so that they can be easily utilized by basic and clinical scientists. NMR isotopomer analysis of small molecule metabolites is well suited to make these measurements because chemical shift and spin-spin coupling allow the isotopomer populations to be very well defined. Knowledge of isotopic distributions in metabolites is tantamount to knowing the flux through the biochemical pathways that generated the isotopomers. The continued focus of Core I is to address technology development for NMR based metabolic analysis with an emphasis on improved sensitivity of NMR isotopomer analysis of in vivo metabolism. The specific aims are: 1. Multiplexing metabolic flux. This aim seeks to expand the number of metabolic pathways measured in a single in vivo experiment by incorporating additional13C tracers of hepatic fluxes into the techniques developed in the previous funding cycle. This aim will improve the experimental efficiency of the NMR isotopomer method (compared to other methods of isotope detection) by making simultaneous measurements of multiple hepatic fluxes. 2. Mathematical analysis of tracer data from complex systems. Our aim is to more fully refine the mathematical models that describe hepatic fluxes with respect to parameter sensitivity, experimental design and extend an existing kinetic model from a simple onecompartment tissue model to a more realistic two-compartment model. This will enhance our capabilities to use 13C NMR multiplet data for analysis of liver and brain metabolism as we move toward more in vivo work on our new high field human scanners. 3. Improve NMR detection of 2H and 13C for metabolic studies. To increase the applicability of our metabolic flux measurements, it is essential that we continue to increase the sensitivity of both the 2H and 13C NMR experiments, the backbone of this methodology. We will extend 2H NMR and JHSQC experiments to 18.8T to take advantage of the increased sensitivity and dispersion of the higher magnetic field and develop the necessary methods to take maximum advantage of new micro-solenoidal probes that offer higher sensitivity for sample limited cases. Finally, we will test the suitability of new high temperature superconducting NMR probes for metabolic measurements.