ISOTOPOMER SPECTRAL ANALYSIS TO QUANTIFY BIOSYNTHESIS

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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.