ISOTOPOMER STUDY OF LIPOGENESIS IN HUMAN HEPATOMA-CELLS IN CULTURE - CONTRIBUTION OF CARBON AND HYDROGEN-ATOMS FROM GLUCOSE

Recent developments in the application of stable isotopes and mass spectrometry have permitted the estimation of precursor enrichment and fractional synthesis of the product through mass isotopomer analysis. Thus, the application of isotopomer analysis in studies with H-2- and C-13-labeled glucose may potentially overcome the limitations of traditional methods which can only estimate the fractional use of carbon and hydrogen from glucose for lipogenesis. To illustrate this approach, isotope incorporation and mass isotopomer distribution were determined in fatty acids and cholesterol from a hepatoma cell line (Hep G2) grown in media containing specific (C1 or C6) 2H- or C-13-labeled glucose. Using the binomial model, the respective precursor enrichment, and fractional synthesis of palmitate, stearate and cholesterol were determined using mass isotopomer distribution analysis. In 1 week, 80% of palmitate, 65.5% of stearate, and 50% of cholesterol molecules in the cell extract were derived from de novo synthesis. Under serum-free condition, glucose contributed about 80% of the carbon of the newly synthesized lipids. Using the relative isotope yield of [1-C-13] and [6-C-13]glucose and a standard formula, the contribution of the pentose pathway to glucose catabolism was calculated to be 4.7%. Fractional syntheses of palmitate, stearate, and cholesterol determined using [1-H-2]glucose agreed well with values determined using C-13-labeled glucose. After correcting for the contribution of deuterium label from the glycolytic pathway, the deuterium from [1-H-2]glucose contributed 4.7% of the total reducing equivalents for lipogenesis. Unlike radioisotope studies, the stable isotope approach provides information from the perspective of the product and insight into the economy of acetyl units and reducing equivalents which were otherwise not available. (C) 1995 Academic Press, Inc.