IN VIVO 13C NMR SPECTROSCOPY

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 这个TR&D项目代表了资源中稳定同位素工作的一个新方向。13 C和2 H示踪剂的新方法和新应用在资源中得到了发展和广泛应用。到目前为止，体内研究的前景在很大程度上限于对人类血液或尿液的检查，以及对实验动物的组织活检。在过去的4年中，UT Southwestern和该资源公司在两项技术上投入了大量资金，即7 T和动态核极化，这使得能够转化为体内光谱学和成像。该项目的目标是进一步开发这些使能技术，以支持当前的临床研究，并为不久的将来的人体研究奠定基础。 目的1研究多重态~（13）C核磁共振波谱在脑柠檬酸循环和多巴胺能流分析中的应用。将开发综合数学工具，用于分析发生在一个以上隔间的大型代谢过程网络。重点将是哺乳动物的大脑。由自旋耦合多重峰提供的13 C NMR谱的高信息含量以前没有被用于多个隔室中的动力学分析。一组微分方程描述所有13 C同位素异构体的所有相关的中间体在神经胶质细胞和神经元已经开发。因此，任何组合的13 C分数富集或13 C多重峰，测量作为时间的函数，可以拟合导出通量。这种方法的优点是减少了对代谢系统的先验假设，提高了代谢变量估计的精度。两个模型正在开发中，是适合于分析的大脑光谱，一个分析随时间收集的光谱（动力学分析），第二个在代谢稳定状态下的单一光谱的分析。该软件将提供给科学界。 目的二是进一步发展7 T磁共振波谱技术，用于脑和骨骼肌代谢的研究。该项目旨在开发和完善7 T光谱学，用于分析人体骨骼肌和大脑中的代谢和生物标志物检测。主要的技术目标是安装2通道并行传输能力，以改善1H光谱和大脑的化学位移成像。脑恶性肿瘤患者的1H MR光谱将与通过活检获得的13 C数据相关。将改进用于骨骼肌13 C NMR光谱的宽带1H去耦，并将向骨骼肌代谢的临床研究者提供包括31 p、13 C和1H光谱的一揽子光谱研究。 目的3研究心肌13 C NMR谱中缺血与底物的相互作用。心肌暴露于[1- 13 C]丙酮酸后，13 C碳酸氢盐/[1- 13 C]乳酸盐的比值对缺血和竞争生理底物（如长链脂肪酸和酮）浓度的变化敏感。我们将测试是否13 C-富集的水溶性短链脂肪酸可用于探测流量的柠檬酸循环的心脏独立的竞争底物。将向离体大鼠心脏提供长链脂肪酸和其他底物的生理混合物。在一系列条件下，通过13 C NMR同位素异构体分析检查短链脂肪酸与生理底物进入乙酰辅酶A库的竞争。将在离体小鼠心脏和大鼠心脏中测试最佳脂肪酸，作为碳1中13 C超极化后柠檬酸循环的合适探针，并检测富含C5的谷氨酸。最后，将针对竞争底物测试最佳分子的体内13 C成像。 目的4探讨超极化~（13）C显像回补的可行性。将碳送入柠檬酸循环用于生物合成目的的反应途径被称为“回补序列”。这些反应在关键的合成过程中起着核心作用，但还没有通用的方法来专门成像这些途径。超极化[U-13 C]乙酸和超极化[U-13 C]烟酸将用于标记离体心脏中谷氨酸的碳4和碳5。由于回补对谷氨酸13 C谱的影响是由于谷氨酸碳3、2和1中13 C富集的变化，触发器光谱（FLOPSY-8）将探测谷氨酸质子化碳中的13 C多重峰，并在几秒钟内测量回补反应的活性。将采用常规同位素异构体方法对该方法进行验证。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This TR&D project represents a substantially new direction for stable isotope work in the Resource. New methods and applications for 13C and 2H tracers have been developed in the Resource and applied widely. Until now the prospect of in vivo studies was limited, for the most part, to examination of blood or urine from humans, and tissue biopsies from experimental animals. Over the last 4 years UT Southwestern and this Resource have invested heavily in two technologies, the 7T and dynamic nuclear polarization, that enables translation to in vivo spectroscopy and imaging. The goal of this project is to further develop these enabling technologies to support current clinical research and to lay the foundation for human studies in the near future. Aim 1 is designed to investigate the utility of multiplet 13C NMR spectroscopy for analysis of citric acid cycle and glutamatergic fluxes in the brain. Integrated mathematical tools for analysis of large networks of metabolic processes occurring in more than one compartment will be developed. The focus will be the mammalian brain. The high information content of 13C NMR spectra provided by spin-coupled multiplets has not previously been used for analysis of kinetics in multiple compartments. A set of differential equations describing all 13C isotopomers of all relevant intermediates in glia and neurons has been developed. Consequently, any combination of 13C fractional enrichment or 13C multiplets, measured as a function of time, can be fit to derive fluxes. The advantage of this approach is fewer prior assumptions about the metabolic system and improved precision in the estimate of metabolic variables. Two models are under development that are suitable for analysis of brain spectra, one to analyze spectra collected over time (kinetic analysis), and a second for the analysis of a single spectrum at metabolic steady state. The software will be made available to the scientific community. Aim 2 will further develop MR spectroscopy at 7T for studies of brain and skeletal muscle metabolism. This project is designed to develop and refine spectroscopy at 7T for analysis of metabolism and detection of biomarkers in human skeletal muscle and brain. The major technical goal is installation of 2-channel parallel transmit capabilities for improved 1H spectroscopy and chemical shift imaging of the brain. 1H MR spectra from patients with brain malignancies will be correlated with 13C data obtained by biopsy. Broad band 1 H decoupling for 13C NMR spectroscopy of skeletal muscle will be refined, and a package of spectroscopy studies including 31p, 13C and 1H spectroscopy will be provided to clinical investigators in skeletal muscle metabolism. Aim 3 examines the interaction of ischemia and substrates in the 13C NMR spectrum of the heart. After exposure of the myocardium to [1-13C]pyruvate, the ratio 13C bicarbonate/[1-13C]lactate is sensitive to ischemia and to changes in the concentration competing physiological substrates such as long chain fatty acids and ketones. We will test whether 13C-enriched water soluble short chain fatty acids can be used to probe flux in the citric acid cycle of the heart independent of competing substrates. Isolated rat hearts will be supplied with physiological mixtures of long chain fatty acids and other substrates. Competition of a short chain fatty acid with physiological substrates for entry into the acetyl-CoA pool will be examined by 13C NMR isotopomer analysiS over a range of conditions. The optimal fatty acid will be tested in isolated mouse hearts and rat hearts as a suitable probe for the citric acid cycle after hyperpolarization of 13C in carbon 1, and detection of glutamate enriched in C5. Finally, the optimal molecule will be tested for 13C imaging in vivo against competing substrates. Aim 4 will investigate the feasibility of imaging anaplerosis with hyperpolarized 13C. Reaction pathways feeding carbon into the citric acid cycle for biosynthetic purposes are termed "anaplerotic sequences". These reactions playa central role in key synthetic processes yet there is no general method for specifically imaging these pathways. Hyperpolarized [U-13C]acetic acid and hyperpolarized [U-13C]pyruvic acid will be used to label carbons 4 and 5 of glutamate in the isolated heart. Since the effects of anaplerosis on the 13C spectrum of glutamate are due to changes in 13C enrichment in carbons 3, 2 and 1 of glutamate, flip-flop spectroscopy (FLOPSY-8) will probe 13C multiplets in protonated carbons of glutamate and measure in a few seconds the activity of anaplerotic reactions. The method will be validated with conventional isotopomer methods.