Use of 13C NMR for Investigation of Ethanol Metabolism in Perfused Liver
Time courses of 13C labels from alanine or lactate and ethanol in perfused mouse livers have been followed by NMR. The enrichment at specific carbons of glucose, glutamate, glutamine, aspartate, acetate, acetoacetate, (β-hydroxybutyrate and lactate has been measured. The specific labeling of glutamate in the presence of labeled alanine and labeled or unlabeled ethanol shows that under these conditions alanine enters the tricarboxylic cycle almost exclusively through pyruvate carboxylation, whereas ethanol is the exclusive source of acetyl-CoA. By comparing the randomization of 13C between C3 and C2 of glutamate it is possible to estimate the mitochondrial fumarase activity; the C6-to-C5 ratios in glucose give the additional scrambling by cytosolic fumarase exchange. These two ratios provide the basis for a continuous study of the activity of the malate-aspartate cycle during the metabolism of ethanol.
Recently we have shown how 13C NMR experiments can be used to determine the intermediates and end products of gluconeogenesis from labeled glycerol in suspensions of isolated rat liver cells (Cohen et al., 1979a). In a single spectrum it was possible to measure the flux through the major pathway, e.g. from [2-13C] glycerol into C2 and CS of glucose, and simultaneously to estimate the pentose cycle activity. The present paper is a report of 13C NMR studies of gluconeogenesis from [3-13C] alanine and from [2-13C] lactate in perfused mouse liver. The competition between ethanol and alanine into the tricarboxylic acid cycle is investigated and a preliminary account of the isotopic approach for studying the importance of the malateaspartate cycle mechanism during the metabolism of ethanol is given. Amore detailed report of the studies involving [3-13C] alanine appears elsewhere (Cohen et al., 1979b).
Male Swiss-Webster mice (25–35g), fasted 24 h, were used. The recirculating perfusion fluid was Krebs bicarbonate buffer containing 3% dialyzed bovine serum albumin and equilibrated with 95% O2/5% CO2 . The perfused liver was positioned in a 15 mm diameter NMR tube; 13C NMR spectra were measured at 35 ± 1°C on a Bruker WH-360 spectrometer at 90.5 MHz.
KeywordsTricarboxylic Acid Cycle Pyruvate Carboxylase Perfuse Liver Ethanol Oxidation Ethanol Metabolism
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- Cohen, S. M., Ogawa, S. and Shulman, R. G., 1979a, 13C NMR studies of gluconeogenesis in rat liver cells: utilization of labeled glycerol by cells from euthroid and hyperthyroid rats, Proc. Natl. Acad. Sci., 76: 1063.Google Scholar
- Grunnet, N. and Katz, J., 1978, Effects of ammonia and norvaline on lactate metabolism by hepatocytes from starved rats, Biochem. J., 172: 595.Google Scholar
- Katz, J. and Rognstad, R., 1967, The labeling of pentose phosphate from glucose-14C and estimation of the rates of transaldolase, transketolase, the contribution of the pentose cycle, and ribose phosphate synthesis, Biochemistry, 2227: 6.Google Scholar
- Krebs, H. H., Lund, P. and Stubbs, M., 1976, Interrelations between gluconeogenesis and urea synthesis in: “Gluconeogenesis: Its Regulation in Mammalian Species,” R. W. Hanson and M. A. Mehlman eds., John Wiley, New York.Google Scholar
- Randle, P. J., 1978, Pyruvate dehydrogenase complex - meticulous regulator of glucose in animals, Trends in Biochem. Sci., 3: 217.Google Scholar
- Utter, M. F. and Scrutton, M. C., 1969, Pyruvate carboxylase, in: “Current Topics in Cellular Regulation,” Vol. 1, B. L. Horecker and E. R. Stadtman, eds., Academic Press, New York.Google Scholar
- Williamson, J. R., Ohkawa, K. and Meijer, A. J., 1974a, Regulation of ethanol oxidation in isolated rate liver cells, in: “Alcohol and Aldehyde Metabolizing Systems,” R. G. Thurman, T. Yonetani, J. R. Williamson and B. Chance, eds., Academic Press, New York.Google Scholar
- Williamson, J. R., Meijer, A. J. and Ohkawa, K., 1974b, Interrelations between anion transport, ureogenesis and gluconeogenesis in isolated rat liver cells, in: “Regulation of Hepatic Metabolism,” F. Lundquist, and N. Tygstrup, eds., Munksgaard, Copenhagen.Google Scholar
- Williamson, J. R., 1976, Role of anion transport in regulation of metabolism, in: “Gluconeogenesis: Its Regulation in Mammalian Species,” R. W. Hanson and M. A. Mehlman, eds., John Wiley, New York.Google Scholar