ISOTOPOMER SPECTRAL-ANALYSIS OF CHOLESTEROL-SYNTHESIS - APPLICATIONS IN HUMAN HEPATOMA-CELLS

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 t he observed spectrum of cholesterol isotopomers with a mathematical mo del to obtain the best fit of model spectrum to data spectrum. The mod el was based on multinomial probability expressions that simulate chol esterol synthesis as a condensation of mevalonate fragments. As many a s four unknown parameters, representing fluxes between compartments, w ere 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 amo unt 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 poly merization and for models with more than two unknown parameters.