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 spe ctrometry have permitted the estimation of precursor enrichment and fr actional synthesis of the product through mass isotopomer analysis. Th us, the application of isotopomer analysis in studies with H-2- and C- 13-labeled glucose may potentially overcome the limitations of traditi onal methods which can only estimate the fractional use of carbon and hydrogen from glucose for lipogenesis. To illustrate this approach, is otope incorporation and mass isotopomer distribution were determined i n 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 fra ctional synthesis of palmitate, stearate and cholesterol were determin ed using mass isotopomer distribution analysis. In 1 week, 80% of palm itate, 65.5% of stearate, and 50% of cholesterol molecules in the cell extract were derived from de novo synthesis. Under serum-free conditi on, glucose contributed about 80% of the carbon of the newly synthesiz ed lipids. Using the relative isotope yield of [1-C-13] and [6-C-13]gl ucose 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]glucos e agreed well with values determined using C-13-labeled glucose. After correcting for the contribution of deuterium label from the glycolyti c pathway, the deuterium from [1-H-2]glucose contributed 4.7% of the t otal reducing equivalents for lipogenesis. Unlike radioisotope studies , the stable isotope approach provides information from the perspectiv e of the product and insight into the economy of acetyl units and redu cing equivalents which were otherwise not available. (C) 1995 Academic Press, Inc.