| I conducted a study coupling metabolomics and mass isotopomer analysis to test the zonation of liver metabolism of gluconeogenic and citric acid cycle intermediates. By comparing the concentration and the labeling of each intermediate, and applying mass isotopomer distribution analysis for the calculation of fractional gluconeogenesis, the metabolic zonation of gluconeogenic and citric acid cycle intermediates in the liver can be identified. For the metabolomic study, I first developed a simple method for tissue extraction of water-soluble intermediates by chloroform-methanol, and obtained the labeling and relative concentration of most of the citric acid cycle and gluconeogenic intermediates in two gas chromatography mass spectrometry runs. This method was applied to my liver perfusion experiments with either NaH13CO 3 or [1,4-13C2]succinate dimethylester. Several heterogeneities were found in these liver perfusions: (i) higher labeling of glyceraldehyde-3-phosphate (GAP) compared to phosphoenolpyruvate (PEP) in NaH13CO3 perfusions, but lower labeling of GAP than PEP in [1,4-13C2]succinate dimethylester perfusions, (ii) lower labeling of PEP than half that of glucose, (iii) labeling difference of GAP to dihydroxyacetone phosphate and carbon 4 to carbon 3 of glucose, (iv) overestimation of fractional gluconeogenesis in some cases calculated from the mass isotopomer distribution of glucose and triose phosphates, and (v) labeling heterogeneity of citric acid cycle intermediates in the effluent and in the liver. These heterogeneities probably result from the zonation of hepatic metabolism and, in some cases, from differences in the labeling pattern of mitochondrial vs extra-mitochondrial metabolites. Overall, my data show that gluconeogenic and citric acid cycle intermediates cannot be considered as sets of homogeneously labeled pools in metabolic studies.; Data on output of citric acid cycle intermediates in the perfused livers showed that alpha-ketoglutarate output ranged from 21-91% of the total output of citric acid cycle intermediates in liver perfusions with lactate, pyruvate or succinate +/- inhibitors. This indicates that alpha-ketoglutarate is a major cataplerotic substrate in some cases without providing extraneous ammonium.; I also demonstrated the reversibility of isocitrate dehydrogenase in intact livers, even without a direct increase of the pool of alpha-ketoglutarate, by comparing the labeling on different carbons of citrate with the fragmentation on gas chromatography mass spectrometry. |
