COMPREHENSIVE ANALYSIS OF METABOLIC FLUXES IN VIVO

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 尽管分子生物学的最新进展为疾病的转录基础提供了更全面的了解，但允许在体内测量转录的最终最终产物代谢通量的技术非常有限。在一些动物模型中，酶表达的变化与通过酶的通量的变化不匹配，这些例子突显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，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能够轻松地使用这些技术。小分子代谢物的核磁共振同位异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异体群体。了解代谢物中的同位素分布等同于通过生物化学途径了解产生同位素异构体的通量。Core I的继续重点是解决基于核磁共振的代谢分析的技术开发，重点是提高体内新陈代谢的核磁共振同位素分析的灵敏度。其具体目标是：1.多路代谢流。这一目标旨在通过将额外的肝脏通量的13C示踪剂纳入上一个资金周期开发的技术来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脏流量来提高核磁共振同位素方法的实验效率(与其他同位素检测方法相比)。2.复杂体系示踪数据的数学分析。我们的目标是从参数敏感性、实验设计和将现有的动力学模型从简单的一室组织模型扩展到更真实的二室模型方面，更全面地改进描述肝脏流量的数学模型。这将增强我们使用13C核磁共振多路数据分析肝脏和大脑新陈代谢的能力，因为我们将在我们的新高场人体扫描仪上进行更多的活体工作。3.改进~2H和~(13)C的核磁共振检测，用于代谢研究。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核磁共振实验的灵敏度，这是这一方法的支柱。我们将把2H核磁共振和JHSQC实验扩展到18.8T，以利用更高磁场的灵敏度和分散性，并开发必要的方法，以最大限度地利用新的微螺线管探头，这些探头在样本有限的情况下提供更高的灵敏度。最后，我们将测试新的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.