Expression and specific activities of carbamoyl phosphate synthetase 1 in chronic hypoxic rats

  • Uly A. Nikmah Biomedical Sciences Graduate Program, Faculty of Medicine, Universitas Indonesia, Jakarta
  • Ani R. Prijanti Department of Biochemistry and Biology Molecular, Faculty of Medicine, Universitas Indonesia, Jakarta
  • Sri W.A. Jusman Department of Biochemistry and Biology Molecular, Faculty of Medicine, Universitas Indonesia, Jakarta
  • Mohamad Sadikin Department of Biochemistry and Biology Molecular, Faculty of Medicine, Universitas Indonesia, Jakarta
Keywords: CPS1, HIF-1, hypoxia, urea biosynthesis
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Abstract

Background: Urea biosynthesis is a very important process in the liver which needs ATP, CO2 and functional mitochondria or aerobic condition. Liver can adapt to hypoxic condition, generally and locally. This study aimed to analyze the effect of chronic hypoxia on liver urea biosynthesis as indicated by the level and specific activity of mRNA of carbamoyl phosphate synthetase 1 (CPS1), a key enzyme in urea biosynthesis in hypoxic rats.

Methods: 20 male Sprague-Dawley rats were placed in hypoxic chamber supplied by a mixture of 10% O2 and 90% N2. Five rats were sacrificed at 1, 3, 5, and 7 days after exposure. Liver homogenates were analyzed for HIF-1 (hypoxia inducible factor-1) by ELISA, CPS1 mRNA by real time RT-PCR and CPS1 enzymatic specific activities by Pierson method. Data were analyzed by ANOVA test and Pearson correlation.

Results: The HIF-1 in liver increased significantly, as well as CPS1 mRNA and CPS1 enzymatic activities (p<0.05). There was a strong correlation (r=0.618; p<0.01) between the level of CPS1 mRNA and CPS1 enzymatic activities, moderate correlation between HIF-1 and CPS1 mRNA (r=0.419; p<0.05) but no correlation between HIF-1 and CPS1 enzymatic activities. The study indicated that urea biosynthesis in liver was affected by hypoxia and partially under HIF-1 regulation. The study also found increase of urea and NH3 biosynthesis related to proteolysis as indicated by the decrease of total body weight and liver weight.

Conclusion: There was an increase in the expression and specific activities of CPS1 in urea biosynthesis as a result of increasing proteolysis in chronic hypoxic condition.

References

  1. Botham KM, Mayes PA. The respiratory chain and oxidative phosphorylation. In Harper's Illustrated Biochemistry, editor. 28th ed. New York: McGrawHill; 2006. p. 103–12.

  2. Nath B, Szabo G. Hypoxia and hypoxia inducible factors: diverse roles in liver diseases. Hepatology. 2012;55(2):622–33. http://dx.doi.org/10.1002/hep.25497

  3. Marks DB, Marks AD, Smith CM. Biokimia kedokteran dasar: sebuah pendekatan klinis. In: Suyono J, Sadikin V, Mandera LI, editors. Basic medical biochemistry: a clinical approach. Jakarta: Penerbit EGC; 1996. p. 701–50. Indonesian.

  4. Pekkala S, Martinez AI, Barcelona B, Gallego J, Bendala E, Yefimenko I, et al. Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase. Biochem J. 2009;424(2):211–20. http://dx.doi.org/10.1042/BJ20090888

  5. Suarez I, Bodega G, Fernandez B. Glutamine synthetase in brain: effect of ammonia. Neurochem Int. 2002;41(2–3):123–42. http://dx.doi.org/10.1016/S0197-0186(02)00033-5

  6. Kurashima K, Hoshi T, Takaku Y, Kanauchi T, Nakamoto K, Ueda M, et al. Changes in the airway lumen and surrounding parenchyma in chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2013;8:523–32. http://dx.doi.org/10.2147/COPD.S52637

  7. Mydin HH, Murphy S, Clague H, Sridharan K, Taylor IK. Anemia and performance status as prognostic markers in acute hypercapnic respiratory failure due to chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis. 2013;8:151–7. http://dx.doi.org/10.2147/COPD.S39403

  8. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001;25(4);402-8. http://dx.doi.org/10.1006/meth.2001.1262

  9. Pierson DL. A rapid colorimetric assay for carbamyl phosphate synthetase 1. J Biochem Biophys Methods. 1980;3(1):31–7. http://dx.doi.org/10.1016/0165-022X(80)90004-4

  10. Harahap, Indriati P. Analysis of blood biochemistry: biochemistry of laboratory experiment. Department of Biochemistry, Faculty of Medicine, Universitas Indonesia. 2001.

  11. Dahlan MS. Statistik untuk kedokteran dan kesehatan: deskripsi, bivariate, multivariat, dilengkapi aplikasi dengan menggunakan SPSS. 5th Ed. Salemba Medika: Jakarta; 2011. 167–88. Indonesian

  12. Semenza GL. Hypoxia-inducible factor-1: oxygen homeostasis and disease pathophysiology. Trends Mol Med. 2001;7(8):345–50. http://dx.doi.org/10.1016/S1471-4914(01)02090-1

  13. Lieberman M, Marks AD. Basic medical biochemistry: a clinical approach. 3rd Ed. Philadelphia: Lippincot Williams and Wilkins; 2009; 697–711.

  14. Adeva MM, Gema S, Natalia B, Donapetry Cristóbal. Ammonium metabolism in humans. Metabolism clinic and experimental. 2012;61:1495–511. http://dx.doi.org/10.1016/j.metabol.2012.07.007

  15. Thoden JB, Xinyi H, Frank M. Carbamoyl-phosphate synthetase: creation of an escape route for ammonia. Journal of Biology Chemical. 2002;277:39722–7. http://dx.doi.org/10.1074/jbc.M206915200

  16. Pekkala S, Martí­nez AI, Barcelona B, Gallego J, bendala E, Yefimenko I, et al. Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase 1. Biochemical J. 2009:424(2);211–20. http://dx.doi.org/10.1042/BJ20090888

  17. Nord FF. Mechanism and regulation of the glutamine-dependent carbamyl phosphate synthetase of Escherichia coli. In: Meister A, editor. Advances in enzymology and related areas of molecular biology. New York: John Wiley and Sons; 1989; 315–74.

  18. Kietzmann T, Cornesse Y, Brechtel K et al. Perivenous Expression of the mRNA of three hypoxia-inducible factor alpha subunits, HIF-1alpha, HIF-2-alpha, HIF-3alpha, in rat liver. Biochem J. 2001; 354: 531–7. http://dx.doi.org/10.1042/bj3540531

  19. Dufour JF, Clavien PA. Signalling pathways in liver disease. 3rd ed. Berlin: Springer; 2005; 442. http://dx.doi.org/10.1007/b138739

  20. Kaluz S, Kaluzová M, Stanbridge EJ. Regulation of gene expression by hypoxia: integration of the HIF transduced hypoxic signal at the hypoxia-responsive element. Clinica Chimica Acta. 2008; 395(1–2): 6–13. http://dx.doi.org/10.1016/j.cca.2008.05.002

  21. Greijer AE, Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1). Journal Pathology. 2005;206: 291–304. http://dx.doi.org/10.1002/path.1778

  22. Nuria P. Hypoxia promotes glycogen accumulation through Hypoxia Inducible Factor (HIF)-mediated induction of glycogen synthase 1. PLoS One. 2010; 5(3): e9644. http://dx.doi.org/10.1371/journal.pone.0009644

  23. Storey, KB. Biochemical adaptation. in: Storey KB (Ed.). Functional metabolism: regulation and adaptation. Wiley-Liss, Inc, Hoboken, New Jersey. 2004; p. 383–413. http://dx.doi.org/10.1002/047167558X

Published
2016-04-15
How to Cite
1.
Nikmah UA, Prijanti AR, Jusman SW, Sadikin M. Expression and specific activities of carbamoyl phosphate synthetase 1 in chronic hypoxic rats. Med J Indones [Internet]. 2016Apr.15 [cited 2024Apr.19];25(1):3-. Available from: http://mji.ui.ac.id/journal/index.php/mji/article/view/1213
Section
Basic Medical Research

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