Cholesterol synthesis from C-13-labeled precursors produces a discrete spectrum of mass isotopomers detectable using gas chromatography-mass spectrometry. The isotopomer spectral analysis (ISA) method matches the observed spectrum of cholesterol isotopomers with a mathematical model to obtain the best fit of model spectrum to data spectrum. The model was based on multinomial probability expressions that simulate cholesterol synthesis as a condensation of mevalonate fragments. As many as four unknown parameters, representing fluxes between compartments, were included in the model. Models were developed to assess cholesterol synthesis from C-13-enriched precursors including mevalonate, acetate, acetoacetate or octanoate. Models were tested in the human hepatoma cell line, Hep G2, which readily incorporated the C-13 substrates into cholesterol. The ISA approach was used to estimate the fractional amount of the cholesterol precursors derived from the C-13 substrate and the fraction of total cellular cholesterol synthesized in the presence of the C-13 substrate. The study demonstrated the feasibility of the ISA approach for a condensation biosynthesis that is not a simple polymerization and for models with more than two unknown parameters.
