ISOTOPOMER SPECTRAL ANALYSIS TO QUANTIFY BIOSYNTHESIS

同位素异构体光谱分析以量化生物合成

• 批准号: 2144386
• 负责人: JOANNE Keene KELLEHER
• 金额: $ 30.54万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-06-01 至 1997-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144386
• 关键词: adipocytes; carbohydrate biosynthesis; cholesterol; fatty acid biosynthesis; gas chromatography mass spectrometry; gluconeogenesis; ketone body; laboratory rat; lipid biosynthesis; liver cells; mathematical model; method development; palmitates; stable isotope; steroid biosynthesis; stoichiometry; triglycerides

Research and treatment of metabolic diseases requires accurate estimates of the rates of biosynthesis of many compounds. Unfortunately, current methods for estimating the rates of synthesis of key compounds in vivo often provide only approximate values and are sometimes unreliable. The overall goal of this project is to examine the potential of a new stable isotope method for quantifying biosynthesis, the "Isotopomer Spectral Analysis" (ISA) method. This method is theoretically applicable for all reactions with a stoichiometry of n A -> 1 B, where n is an integer greater than 1. (Examples include, 8 acetate -> 1 palmitic acid and 2 lactate -> glucose). If "A" is labelled with 13C, mass isotope variants (isotopomers) of the product "B" appear. The ISA method is dependent on measurements of the relative abundance of the isotopomers using gas chromatography/ mass spectrometry operating in the specific ion monitoring mode (GC/MS SIM) and on a mathematical model of the biosynthetic reaction under study. The mathematical model produces an independent equation for each mass isotopomer based on the multinomial distribution. Each equation contains two unknown parameters related to the rate of synthesis of the product B. The parameters are: the fractional contribution of the tracer to the intracellular precursor pool for biosynthesis (D); and, the fraction of the sampled compartment of B derived from the tracer enriched biosynthetic reaction (G). To estimate these parameters, a nonlinear least squares procedure is used. The four specific aims of this project are to develop and test this method for the synthesis of four important metabolites, fatty acids (palmitate), ketone bodies (acetoacetate) cholesterol, and glucose. The proposed studies range from relatively simple in vitro validation protocols to in vivo feasibility tests and theoretical analyses. This range of experiments was selected to provide a thorough examination of the potential of the ISA method. Parameter estimates will be assessed in terms of goodness of fit, model and measurement error. The long term goal of the project is to develop and utilize this now method to assess biosynthetic reactions in man. However, before the technique can be profitably applied to human subjects, studies must be carried out under carefully controlled conditions to determine the optimal conditions and limits of the method. The development and application of the ISA method is particularly promising for research and treatment related to the metabolic alterations in Diabetes Mellitus.

代谢性疾病的研究和治疗需要准确的估计 许多化合物的生物合成速度。不幸的是，目前 体内关键化合物合成速率的估算方法 通常只提供近似值，有时并不可靠。这个 这个项目的总体目标是检查一个新的稳定的潜力 定量生物合成的同位素法--“同质异构体光谱” (ISA)方法。该方法在理论上适用于所有 化学计量比为n A-1 B的反应，其中n为大于1的整数 (实例包括8-乙酸酯-1-棕榈酸和2-乳酸-GT； 葡萄糖)。如果“A”标有13C，质量同位素变种(同位素异构体) 的产品“B”出现了。ISA方法取决于以下测量 用气相色谱/质量法测定同分异构体的相对丰度 在特定离子监测模式(GC/MS SIM)下运行的光谱和 关于正在研究的生物合成反应的数学模型。这个 数学模型为每个质量产生一个独立的方程 基于多项式分布的同分异构体。每个方程式都包含 与产物B的合成速度有关的两个未知参数。 这些参数是：示踪剂对 细胞内生物合成前体池(D)；以及 从示踪剂富集型生物合成物中提取的B的取样室 反应(G)。为了估计这些参数，非线性最小二乘法 使用的是程序。这个项目的四个具体目标是开发 并测试这种方法合成四种重要的代谢物脂肪 酸(棕榈酸)、酮体(乙酰乙酸酯)、胆固醇和葡萄糖。 建议的研究范围从相对简单的体外验证 体内可行性测试和理论分析的协议。这 选择了一系列实验，以提供对 ISA方法的潜力。参数估计将按下列条件进行评估 拟合优度、模型和测量误差。中国的长期目标是 项目是开发和利用这种NOW方法来评估生物合成 人类的反应。然而，在这项技术能够有利可图地应用之前 对于人类受试者，研究必须在仔细控制的情况下进行 确定了该方法的最佳条件和限度。 ISA方法的发展和应用特别有前景 与糖尿病代谢改变相关的研究和治疗 糖尿病。