Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Nov 1;287(Pt 3):813–820. doi: 10.1042/bj2870813

Real-time study of the urea cycle using 15N n.m.r. in the isolated perfused rat liver.

A Geissler 1, K Kanamori 1, B D Ross 1
PMCID: PMC1133080  PMID: 1445243

Abstract

1. Isolated rat liver was perfused with 10 mM-15NH4Cl, 5 mM-lactate and 1 mM-ornithine, or with 3 mM-[15N]alanine and 1 mM-ornithine, in haemoglobin-free medium. The liver was physiologically stable for over 3 h and synthesized urea at the rate of 1.15 mumol.min-1.g of liver-1 (15NH4(+)-perfused) or 0.41 mumol.min-1.g-1 ([15N]alanine-perfused). 2. The perfused liver was continuously monitored by 15N n.m.r. spectroscopy at 20.27 MHz for 15N. Well-resolved 15N resonances of precursors and intermediates of the urea cycle, present at tissue concentrations of 0.2-3.0 mumol/g, were observed from the intact liver in 5-40 min of acquisition. Key metabolites in liver extract and the final perfusion medium were analysed by n.m.r. and by biochemical assays to determine fractional 15N enrichment and the total 15N recovery. 3. In 15NH4(+)-perfused liver (n = 6), 15N incorporation into glutamate and alanine (1.0-1.3 mumol/g), as well as progressive formation of [15N2]urea, was observed during the first 2 h of perfusion. In the second and third hour, hepatic concentrations of [omega-15N]citrulline and [omega,omega'-15N]argininosuccinate increased to n.m.r.-detectable levels (0.3-0.9 mumol/g). The [15N]aspartate pool was large in the absence of added ornithine, but on its addition was rapidly incorporated into argininosuccinate (n = 3). 4. In [15N]alanine-perfused liver, major metabolites were [15N]glutamate, [gamma-15N]glutamine and [15N]urea. Urea-cycle intermediates were undetectable. 5. The results suggest that, in intact liver provided with excess ammonia, low concentrations of cytosolic argininosuccinate synthetase and argininosuccinate lyase limited the rate of metabolite flux in the urea cycle. By contrast, in alanine-perfused liver at a physiological rate of urea synthesis, mitochondrial carbamoylphosphate synthetase was rate-limiting. 6. The potential utility of 15N n.m.r. for study of metabolite channelling through urea-cycle enzymes in intact liver is discussed.

Full text

PDF
813

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brosnan J. T., Williamson D. H. Mechanisms for the formation of alanine and aspartate on rat liver in vivo after administration of ammonium chloride. Biochem J. 1974 Mar;138(3):453–462. doi: 10.1042/bj1380453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. CERIOTTI G., SPANDRIO L. A spectrophotometric method for determination of urea. Clin Chim Acta. 1963 Mar;8:295–299. doi: 10.1016/0009-8981(63)90171-2. [DOI] [PubMed] [Google Scholar]
  3. Chamalaun R. A., Tager J. M. Nitrogen metabolism in the perfused rat liver. Biochim Biophys Acta. 1970 Oct 27;222(1):119–134. doi: 10.1016/0304-4165(70)90357-0. [DOI] [PubMed] [Google Scholar]
  4. Cheung C. W., Cohen N. S., Raijman L. Channeling of urea cycle intermediates in situ in permeabilized hepatocytes. J Biol Chem. 1989 Mar 5;264(7):4038–4044. [PubMed] [Google Scholar]
  5. Cohen N. S., Kyan F. S., Kyan S. S., Cheung C. W., Raijman L. The apparent Km of ammonia for carbamoyl phosphate synthetase (ammonia) in situ. Biochem J. 1985 Jul 1;229(1):205–211. doi: 10.1042/bj2290205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooper A. J., Nieves E., Coleman A. E., Filc-DeRicco S., Gelbard A. S. Short-term metabolic fate of [13N]ammonia in rat liver in vivo. J Biol Chem. 1987 Jan 25;262(3):1073–1080. [PubMed] [Google Scholar]
  7. Cooper A. J., Nieves E., Rosenspire K. C., Filc-DeRicco S., Gelbard A. S., Brusilow S. W. Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver. J Biol Chem. 1988 Sep 5;263(25):12268–12273. [PubMed] [Google Scholar]
  8. Cunningham C. C., Malloy C. R., Radda G. K. Effect of fasting and acute ethanol administration on the energy state of in vivo liver as measured by 31P-NMR spectroscopy. Biochim Biophys Acta. 1986 Jan 23;885(1):12–22. doi: 10.1016/0167-4889(86)90033-9. [DOI] [PubMed] [Google Scholar]
  9. Elliott K. R., Tipton K. F. Kinetic studies of bovine liver carbamoyl phosphate synthetase. Biochem J. 1974 Sep;141(3):807–816. doi: 10.1042/bj1410807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Farrow N. A., Kanamori K., Ross B. D., Parivar F. A 15N-n.m.r. study of cerebral, hepatic and renal nitrogen metabolism in hyperammonaemic rats. Biochem J. 1990 Sep 1;270(2):473–481. doi: 10.1042/bj2700473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hems R., Ross B. D., Berry M. N., Krebs H. A. Gluconeogenesis in the perfused rat liver. Biochem J. 1966 Nov;101(2):284–292. doi: 10.1042/bj1010284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Häussinger D. Hepatocyte heterogeneity in glutamine and ammonia metabolism and the role of an intercellular glutamine cycle during ureogenesis in perfused rat liver. Eur J Biochem. 1983 Jun 15;133(2):269–275. doi: 10.1111/j.1432-1033.1983.tb07458.x. [DOI] [PubMed] [Google Scholar]
  13. Häussinger D., Weiss L., Sies H. Activation of pyruvate dehydrogenase during metabolism of ammonium ions in hemoglobin-free perfused rat liver. Eur J Biochem. 1975 Apr 1;52(3):421–431. doi: 10.1111/j.1432-1033.1975.tb04010.x. [DOI] [PubMed] [Google Scholar]
  14. Jungermann K., Katz N. Functional specialization of different hepatocyte populations. Physiol Rev. 1989 Jul;69(3):708–764. doi: 10.1152/physrev.1989.69.3.708. [DOI] [PubMed] [Google Scholar]
  15. Kanamori K., Ross B. D., Farrow N. A., Parivar F. A 15N-NMR study of isolated brain in portacaval-shunted rats after acute hyperammonemia. Biochim Biophys Acta. 1991 Jun 5;1096(4):270–276. doi: 10.1016/0925-4439(91)90062-e. [DOI] [PubMed] [Google Scholar]
  16. Legerton T. L., Kanamori K., Weiss R. L., Roberts J. D. 15N NMR studies of nitrogen metabolism in intact mycelia of Neurospora crassa. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1495–1498. doi: 10.1073/pnas.78.3.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ohtake Y., Clemens M. G. Interrelationship between hepatic ureagenesis and gluconeogenesis in early sepsis. Am J Physiol. 1991 Mar;260(3 Pt 1):E453–E458. doi: 10.1152/ajpendo.1991.260.3.E453. [DOI] [PubMed] [Google Scholar]
  18. Osawa T., Tsuji T. Fractionation and structural assessment of oligosaccharides and glycopeptides by use of immobilized lectins. Annu Rev Biochem. 1987;56:21–42. doi: 10.1146/annurev.bi.56.070187.000321. [DOI] [PubMed] [Google Scholar]
  19. Radda G. K. The use of NMR spectroscopy for the understanding of disease. Science. 1986 Aug 8;233(4764):640–645. doi: 10.1126/science.3726553. [DOI] [PubMed] [Google Scholar]
  20. Ratner S. Determination of arginnosuccinate in normal blood serum and liver. Anal Biochem. 1975 Jan;63(1):141–155. doi: 10.1016/0003-2697(75)90198-0. [DOI] [PubMed] [Google Scholar]
  21. Ratner S. Enzymes of arginine and urea synthesis. Adv Enzymol Relat Areas Mol Biol. 1973;39:1–90. doi: 10.1002/9780470122846.ch1. [DOI] [PubMed] [Google Scholar]
  22. Raushel F. M., Garrard L. J. A positional isotope exchange study of the argininosuccinate lyase reaction. Biochemistry. 1984 Apr 10;23(8):1791–1795. doi: 10.1021/bi00303a032. [DOI] [PubMed] [Google Scholar]
  23. Ross B. D., Radda G. K., Gadian D. G., Rocker G., Esiri M., Falconer-Smith J. Examination of a case of suspected McArdle's syndrome by 31P nuclear magnetic resonance. N Engl J Med. 1981 May 28;304(22):1338–1342. doi: 10.1056/NEJM198105283042206. [DOI] [PubMed] [Google Scholar]
  24. Shulman R. G., Brown T. R., Ugurbil K., Ogawa S., Cohen S. M., den Hollander J. A. Cellular applications of 31P and 13C nuclear magnetic resonance. Science. 1979 Jul 13;205(4402):160–166. doi: 10.1126/science.36664. [DOI] [PubMed] [Google Scholar]
  25. Wanders R. J., Van Roermund C. W., Meijer A. J. Analysis of the control of citrulline synthesis in isolated rat-liver mitochondria. Eur J Biochem. 1984 Jul 16;142(2):247–254. doi: 10.1111/j.1432-1033.1984.tb08278.x. [DOI] [PubMed] [Google Scholar]
  26. Williamson D. H., Lund P., Krebs H. A. The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J. 1967 May;103(2):514–527. doi: 10.1042/bj1030514. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES