| Methods were developed for the measurement of absolute metabolite concentrations, as well as metabolic fluxes in cultured cells. Absolute metabolite concentrations were determined using isotopically labeled cell extracts and unlabeled standards. Flux determination was performed by monitoring the dynamic incorporation of stable-isotope labeled compounds along with knowledge of the metabolite concentrations. Both approaches used the measurement of isotopomer ratios, monitored by a liquid chromatograph tandem mass spectrometry approach. The concentrations of over one hundred intracellular metabolites in the model organism Escherichia coli were determined. These concentrations were used to evaluate the expected thermodynamics and kinetics of reaction based upon the previously known free energy of reactions and binding constants of enzymes, finding that the majority of enzyme active sites were saturated for substrate during exponential growth.;During Human Cytomegalovirus (HCMV) infection, human fibroblasts significantly alter their metabolism. In order to quantify these changes, approximately 30 intracellular concentrations were determined, and a battery of experiments monitoring the rate of isotope incorporation were performed. By fitting a series of differential equations to these data, quantitative estimates of cellular metabolic fluxes in both HCMV infected and uninfected cells were determined. Metabolic flux into fatty acid biosynthesis was found to be significantly increased during infection, and inhibition of this pathway by small molecules significantly reduced the ability of HCMV to reproduce. |
