Isotopolog perturbation techniques for metabolic networks: metabolic recycling of nutritional glucose in Drosophila melanogaster.

Drosophila melanogaster strain Oregon-R(*) was grown on standard medium supplemented with [U-(13)C(6)]glucose. One to two days after hatching, flies were extracted with water. Glucose was isolated chromatographically from the extract and was analyzed by (13)C NMR spectroscopy. All (13)C signals of the isolated glucose were multiplets arising by (13)C(13)C coupling. Based on a comprehensive analysis of the coupling constants and heavy isotope shifts in glucose, the integrals of individual (13)C signal patterns afforded the concentrations of certain groups of (13)C isotopologs. These data were deconvoluted by a genetic algorithm affording the abundances of all single-labeled and of 15 multiply labeled isotopologs. Among the latter group, seven isotopologs were found at concentrations >0.1 mol % with [1,2-(13)C(2)]glucose as the most prominent species. The multiply (13)C-labeled glucose isotopologs are caused by metabolic remodeling of the proffered glucose via a complex network of catabolic and anabolic processes involving glycolysis and/or passage through the pentose phosphate, the Cori cycle and/or the citrate cycle. The perturbation method described can be adapted to a wide variety of experimental systems and isotope-labeled precursors.