STUDIES OF CARDIAC METABOLISM BY 13C AND 31P NMR

该项目的主要目标是利用最新的进展 在31 p和13 C核磁共振（NMR）领域取得的进展 光谱研究灌流大鼠心脏的能量学和代谢 在不同的基板供应和工作条件下。 重点将 使用富含13 C的底物来研究 柠檬酸循环，它与苹果酸-柠檬酸循环的相互作用， 柠檬酸循环净池大小增减的机理 中间体的 将使用两个完整心脏获得13 C NMR光谱 和高氯酸提取物。 后者也将用于酶促反应。 测定代谢物以辅助共振的鉴定和定量 从来自13 C光谱的单个13 C标记的碳原子获得。 从13 C特异性标记模式提供的信息， 中间体，以及他们的总池大小的知识， 通过代谢分析确定，将用于构建一个通量模型， 柠檬酸循环和相关的转氨步骤。 模型 将被用来测试各种假设有关的流量调节， 柠檬酸循环在不同底物供给条件下， 心脏在稳态和过渡期间的功输出 循环流量不均匀时的状态。 以下主要具体目标 将得到解决。 1. 确定了反应器中的速率控制步骤 柠檬酸循环和酶抑制剂控制强度的评估 滴定研究。 2. 存在和代谢的评估 代谢物区室化和底物通道化的意义。 3. 通过苹果酸-苹果酸循环的不同通量率对 通过柠檬酸合成酶，α-酮戊二酸的流量调节 脱氢酶和谷氨酸氨基转移酶。 4. 调查 负责引起组织内容物净变化的机制 柠檬酸循环中间体。 5. 乙酰乙酸在 存在和不存在丙酸盐对通过琥珀酸硫激酶的通量的影响， 调节柠檬酸从α-酮戊二酸到苹果酸的循环。 6. 柠檬酸循环功能的研究 缺血后心脏

The major goal of this project is to take advantage of the recent advances made in the field of 31p and 13C nuclear magnetic resonances (NMR) spectroscopy to study the energetics and metabolism of perfused rat hearts under different conditions of substrate supply and work. Emphasis will be given to the use of 13C-enriched substrates to study regulation of the citric acid cycle, its interactions with the malate-asparate cycle and mechanisms for increasing or decreasing net pool sizes of citric acid cycle intermediates. 13C NMR spectra will be obtained using both intact hearts and perchloric acid extracts. The latter will also be used for enzymatic assay of metabolites to aid identification and quantitation of resonances obtained from individual 13C-labeled carbon atoms from the 13C spectra. The information provided from 13C specific labeling patterns of intermediates, together with knowledge of their total pool sizes as determined by metabolic analyses, will be used to construct a flux model of the citric acid cycle and interrelated transamination steps. The model will be used to test various hypotheses concerning regulation of flux in the citric acid cycle under different conditions of substrate supply and work output of the heart during the steady state as well as transition states when cycle flux is nonuniform. The following major specific aims will be addressed. 1. Identification of rate-controlling steps in the citric acid cycle and assessment of enzyme control strengths by inhibitor titration studies. 2. Assessment of the existence and metabolic significance of metabolite compartmention and substrate channeling. 3. Effects of varying flux rates through the malate-asparate cycle on the regulation of flux through citrate syntase, Alpha-ketoglutarate dehydrogenase and asparate aminotransferase. 4. Investigation of the mechanisms responsible for causing net changes of the tissue contents of citric acid cycle intermediates. 5. Effects of acetoacetate in the absence and presence of propionate on flux through succinate thiokinase and regulation of the citric acid cycle from Alpha-keto-glutarate to malate. 6. Investigation of citric acid cycle function in reoxygenated post-ischemic hearts.