GLUCONEOGENESIS IN ISOLATED RAT HEPATOCYTES EVALUATED BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY USING DEUTERATED WATER

Tritiated water and radioactive tracers have been used to monitor gluc ose production by primary cultures of hepatocytes. More recently, (H2O )-H-3 has been replaced for by (H2O)-H-2 in 'in vivo' studies addresse d at the evaluation of the relative contribution of gluconeogenesis to total glucose production. In this work, the possibility of using 2H(2 )O to determine the ratio between the glucogenic flux and the overall flux through glucose 6-phosphate in isolated liver cells in vitro was evaluated. For this purpose, hepatocytes from either fasted or fed rat s were incubated with a medium containing 6, 12 and 25% of 2H(2)O in t he presence of either 2 or 20 mM pyruvate. Isotopomer analysis of six different mass clusters (m/z 328, 314, 242, 212, 187 and 145) was carr ied out by gas chromatography mass spectrometry (GC/MS) of glucose ald onitrile pentaacetate. For each cluster, ions at m/z + 1, +2, +3 and 4 were monitored. From the combination of different clusters the enric hment at C-6 and C-2 of glucose was computed and the C-6/C-2 ratio was considered to represent the contribution of gluconeogenesis to total glucose production, as suggested previously. Based on the results obta ined, conditions selected to be optimum for the use of the method in s tudies on the modulation of gluconeogenesis were as follows: incubatio n of hepatocytes with 20 mM pyruvate in 12% (H2O)-H-2 followed by GC/e lectron ionization MS analysis of the clusters of ions at m/z 328, 314 and 187 of the glucose derivative to calculate enrichment at the C-2 and C-6 positions of glucose. (C) 1998 John Wiley & Sons, Ltd.