NEUROTRANSMITTER FLUXES BY MULTIPLET 13C NMR

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。 目前谷氨酸-谷氨酰胺循环模型中体现的脑代谢的双室描述（1）现在被广泛接受，并且是几个研究组现在常规使用的代谢模型的基础，以确定动物和人体内谷氨酸神经递质的释放速率。简单地说，大脑中的区室化被认为涉及星形胶质细胞和神经元。星形胶质细胞摄取神经元释放的谷氨酸，但具有更高的谷氨酰胺水平以及更高的谷氨酰胺合成酶和丙酮酸羧化酶活性。“谷氨酰胺-谷氨酸”循环通过神经元细胞摄取谷氨酰胺完成，神经元细胞含有大部分谷氨酸并具有较高的谷氨酰胺酶活性。神经胶质中的高丙酮酸羧化酶活性被认为维持柠檬酸循环中间体，并允许碳从星形胶质细胞流向神经元。相比之下，葡萄糖在神经元中的主要命运被认为是通过丙酮酸脱氢酶氧化。 尽管普遍同意的作用，代谢区室在中枢神经系统中，目前的代谢模型用于定量谷氨酸循环作出重大的假设，其中一些正确性仍未解决。13 C NMR提供了一个独特的机会来测试体内不同区室化模型的详细假设，虽然已经取得了相当大的进展，但这些实验提供的大部分信息-特别是同位素异构体标记动力学-目前仍未得到利用。同位素异构体分析使研究人员不仅可以确定特定碳位置的富集动力学，而且可以确定同一分子中紧邻碳位置的富集动力学。每个C位可能出现四种标记模式，并且通常可以区分所有四种所谓的多重态，因此可以从一个碳进行四次测量，而不仅仅是一次。每个测量值代表一组不同的同位素异构体，因此有四个数学上不同的变量，可以通过为模型提供额外的约束来改善路径通量的估计。在动物和人类受试者中已经证明了这种测量是可能的;仍然需要提供新的模型来利用这些额外的信息。 总之，挑战在于了解有关13 C在灵长类动物大脑中分布的所有可用信息。具体来说，我们需要能够解释位点特异性富集信息和多重峰数据，以及两者的任何组合。西南大学的研究资源已经开发了数学工具来做到这一点。这些工具需要在范德比尔特实施，以服务于我们的科学目标。 该研究项目的技术目标是在[1，6 - 13 C]葡萄糖输注过程中获取人脑中谷氨酸盐和其他神经递质的质子去耦13 C NMR光谱，并利用将同位素异构体信息纳入分析的模型提供的额外信息。将这些分析与使用传统二室模型获得的结果进行比较。高优先级是获得具有足够的化学位移分辨率的数据，使得13C_13C自旋-自旋耦合在神经递质如谷氨酸和γ-氨基丁酸中容易分辨。生物学的目标是测量柠檬酸循环的流量和回补反应的速率，包括大脑中神经递质的合成。范德比尔特小组将专注于脉冲序列开发和数据采集; UTSWMC小组将负责分析个体光谱和数据集，并将数据拟合到实验室已经开发的脑代谢双室模型中。 在UT西南，一般两室模型将根据范德比尔特常规获得的数据进行完善。最初，预计自旋耦合多重态数据将可从谷氨酸获得。根据其他数据的可用性，如γ-氨基丁酸、天冬氨酸或谷氨酰胺的富集情况，将对模型进行完善。UT西南小组的主要职责是对范德比尔特提供的13 C光谱进行详细分析，以评估和验证两室模型的假设。特别是，纳入同位素动力学分析将提供独特的机会，交叉验证模型的标签流内和之间的隔室。这些数据最初将用于评估Oz et al模型在恒河猴研究中的性能，随后用于评估人类研究。在整个过程中，将仔细注意模型的适用性。数据和拟合时间曲线之间的差异可能需要重新评估模型。将采取步骤确定是否所有模型参数都是可识别的。还将检查数据中误差的影响，并确定估计参数的置信限。该模型可以适应于创建不同的版本，适合关于大脑区室化的各种假设，并测试这些是否可以通过实验区分。

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. A two-compartment description (1) of brain metabolism embodied in current models of glutamate - glutamine cycling, is now well accepted, and is the basis for the metabolic models now used routinely by several groups to determine the rates of glutamate neurotransmitter release in animals and humans in vivo. Briefly, compartmentation in the brain is thought to involve astrocytes and neurons. Astrocytes take up the glutamate released from neurons, but have higher glutamine levels together with higher glutamine synthetase and pyruvate carboxylase activity. The "glutamine-glutamate" cycle is completed by glutamine uptake by neuronal cells, which contain the majority of glutamate and have higher glutaminase activity. The high pyruvate carboxylase activity in glia is thought to maintain citric acid cycle intermediates and allow the flow of carbon from astrocytes to neurons. In contrast, the main fate of glucose in neurons is thought to be oxidation via pyruvate dehydrogenase. Despite general agreement on the role of metabolic compartmentation in the CNS, current metabolic models used for quantifying glutamate cycling make significant assumptions, some of whose correctness remain unresolved. 13C NMR offers a unique opportunity to test the detailed assumptions of different compartmentation models in vivo, and while there has been considerable progress, much of the information available from these experiments - particularly isotopomer labeling kinetics - remains unexploited at this time. Isotopomer analysis allows the investigator to determine not only the enrichment kinetics of a particular carbon position, but also that of its immediate neighbors in the same molecule. Four possible labeling patterns can arise for each C position, and it is often possible to distinguish all four of these so-called multiplets, and hence from one carbon four measurements can be made, not just one. Each measurement represents a distinct group of isotopomers, and hence four mathematically distinct variables that can improve the estimation of pathway fluxes by providing additional constraints to the model. That such measurements are possible has been demonstrated, both in animals and human subjects; it remains to provide new models to take advantage of this additional information. In summary, the challenge is to understand all the available information about distribution of 13C in the primate brain. Specifically, we need to be able to interpret site-specific enrichment information and multiplet data, and any combination of the two. The Research Resource at Southwestern has developed the mathematical tools to do just that. These tools need to be implemented at Vanderbilt to serve our scientific goals. The technical goal of this research project is to acquire proton-decoupled 13C NMR spectra of glutamate and other neurotransmitters in the human brain during infusion of [1,6-13C] glucose, and to exploit the additional information available from models that incorporate isotopomer information into the analysis. These analyses will be compared with the results obtained using traditional two-compartment models. A high priority is to acquire data with sufficient chemical shift resolution that 13C_13C spin-spin coupling is easily resolved in neurotransmitters such as glutamate and gamma-aminobutyric acid. The biological goal is to measure flux in the citric acid cycle and the rate of anaplerotic reactions, including neurotransmitter synthesis, in the brain. The Vanderbilt group will focus on pulse sequence development and data acquisition; the UTSWMC group will be responsible for analysis of individual spectra and data sets, and for fitting the data to the two-compartment model of brain metabolism already under development in the lab. At UT Southwestern, the general two-compartment model will be refined depending on the data obtained routinely at Vanderbilt. Initially, it is anticipated that spin-coupled multiplet data will be available from glutamate. Depending on the availability of other data such as enrichment in gamma aminobutyric acid, aspartate or glutamine, the model will be refined. The primary responsibility of the UT Southwestern group will be to perform detailed analysis of 13C spectra provided by Vanderbilt, to assess and validate the assumptions of the two compartment model. In particular, incorporation of isotopomer kinetic analyses will provide unique opportunities for cross validation of models of label flow within and between compartments. These data will initially be used to assess the performance of the model of Oz et al in rhesus monkey studies, and subsequently in humans. Throughout this process, careful attention will be given to the suitability of the model. Discrepancies between data and fitted time curves may require re-evaluation of the model. Steps will be taken to determine whether all model parameters are identifiable. The effect of error in the data will also be checked, and confidence limits on estimated parameters determined. The model can be adapted to create different versions that fit with the various hypotheses regarding cerebral compartmentation and test whether these can be distinguished experimentally.