Ways to Increase the Activity of Glutamate Dehydrogenase in Erythrocyte-Bioreactors for the Ammonium Removal

  • D. V. Borsakova
  • E. S. Protasov
  • S. V. Nazarenko
  • Y. G. Alexandrovich
  • A. A. Butylin
  • F. I. Ataullakhanov
  • E. I. SinauridzeEmail author


An increased blood ammonium concentration (hyperammonemia) is toxic to the central nervous system, so removing excess ammonium from the bloodstream is an important task. One of the solutions may be the use of erythrocyte-bioreactors (EBRs) with ammonium-processing enzymes loaded inside. Earlier attempts have been made to create such EBRs based on glutamate dehydrogenase (GDH) or glutamine synthetase; however, these EBRs were not effective enough. We have previously shown that the reasons for this were the low permeability of the erythrocyte membrane for the substrates of these reactions (α-ketoglutarate and glutamate) and the low activity of the included GDH (due to its high molecular mass and tendency towards aggregation at an increase in concentration above 0.1 mg/mL), and also that the low membrane permeability problem for α-ketoglutarate and glutamate could be overcome if GDH and alanine aminotransferase were included into EBR together, because these metabolites should be consumed and reproduced within such EBR cyclically. However, the problem of low GDH activity remains if GDH is the main ammonium processing enzyme. To improve the efficiency of GDH incorporation into erythrocytes and the quality of the EBRs (their erythrocyte indices and osmotic fragility), these parameters were compared for various hypoosmotic methods for loading GDH into cells. In addition, a comparison was made of previously used GDH from bovine liver and GDH from Proteus sp. It was shown that the method of flow dialysis was the most effective and allowed the inclusion of the GDH amount 3 times exceeding the inclusion achieved during the hypoosmotic dialysis. The properties of erythrocytes after exposure to this method changed slightly. GDH from Proteus sp. did not aggregate with an increase in its concentration and thereby allowed an approximately 18-fold increase in the specific activity of the enzyme in erythrocytes. Thus, this GDH is a promising enzyme for creating effective EBRs for ammonium removing.


erythrocyte-bioreactor glutamate dehydrogenase from bovine liver glutamate dehydrogenase from Proteus sp. hyperammonemia hypoosmotic dialysis in the flow 



The work was supported by the fundamental research program Fundamental bases of physiological adaptations technology of the Presidium of the Russian Academy of Sciences.


Conflict of interests. The authors declare that they have no conflict of interest.

Statement of compliance with standards of research involving humans as subjects. The study was approved by the Ethical Committee of the Center for Theoretical Problems of Physicochemical Pharmacology. All participants provided written informed consent before blood collection. The blood of healthy donors was received at the station of blood transfusion and was used without authentication.


  1. 1.
    Bax B.E., Bain M.D., Ward C.P., Fensom A.H., Chalmers R.A. 1997. The entrapment of mannose-terminated glucocerebrosidase (alglucerase) in human carrier erythrocytes. In: Erythrocytes as drug carriers in medicine. Eds Sprandel U., Way J.L. New York. London: Plenum Press USA, p. 59–62.Google Scholar
  2. 2.
    Rossi L., Bianchi M., Magnani M. 2018. Increased glucose metabolism by enzyme-loaded erythrocytes in vitro and in vivo normalization of hyperglycemia in diabetic mice. Biotechnol. Appl. Biochem. 15, 207–216.Google Scholar
  3. 3.
    Kravtzoff R., Desbois I., Lamagnere J.P., Muh J.P., Valat C., Chassaigne M., Colombat P., Ropars C. 1996. Improved pharmacodynamics of L-asparaginase-loaded in human red blood cells. Eur. J. Clin. Pharmacol. 49, 465–470.CrossRefPubMedGoogle Scholar
  4. 4.
    Sanz S., Lizano C., Luque J., Pinilla M. 1999. In vitro and in vivo study of glutamate dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis procedure. Life Sci. 65, 2781–2789.CrossRefPubMedGoogle Scholar
  5. 5.
    Auron A., Brophy P.D. 2012. Hyperammonemia in review: Pathophysiology, diagnosis, and treatment. Pediatr. Nephrol. 27, 207–222.CrossRefPubMedGoogle Scholar
  6. 6.
    Husson M.C., Schiff M., Fouilhoux A., Cano A., Dobbelaere D., Brassier A., Mention K., Arnoux J.B., Feillet F., Chabrol B., Guffon N., Elie C., de Lonlay P. 2016. Efficacy and safety of i.v. sodium benzoate in urea cycle disorders: A multicentre retrospective study. Orphanet J. Rare Dis. 11, 127. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sushma S., Dasarathy S., Tandon R.K., Jain S., Gupta S., Bhist M.S. 1992. Sodium benzoate in the treatment of acute hepatic encephalopathy: A double-blind randomized trial. Hepatology. 16, 138–144.CrossRefPubMedGoogle Scholar
  8. 8.
    Honda S., Yamamoto K., Sekizuka M., Oshima Y., Nagai K., Hashimoto G., Kaneko H., Tomomasa T., Konno Y., Horiuchi R. 2002. Successful treatment of severe hyperammonemia using sodium phenylacetate powder prepared in hospital pharmacy. Biol. Pharm. Bull. 25, 1244–1246.CrossRefPubMedGoogle Scholar
  9. 9.
    Sharma B.C., Sharma P., Lunia M.K., Srivastava S., Goyal R., Sarin S.K. 2013. A randomized, double-blind, controlled trial comparing rifaximin plus lactulose with lactulose alone in treatment of overt hepatic encephalopathy. Am. J. Gastroenterol. 108, 1458–1463.CrossRefPubMedGoogle Scholar
  10. 10.
    Kircheis G., Nilius R., Held C., Berndt H., Buchner M., Görtelmeyer R., Hendricks R., Krüger B., Kuklinski B., Meister H., Otto H.J., Rink C., Rösch W., Stauch S. 1997. Therapeutic efficacy of L-ornithine-L-aspartate infusions in patients with cirrhosis and hepatic encephalopathy: Results of a placebo-controlled, double-blind study. Hepatology. 25, 1351–1360.CrossRefPubMedGoogle Scholar
  11. 11.
    Protasov E.S., Borsakova D.V., Alexandrovich Y.G., Korotkov A.V., Kosenko E.A., Butylin A.A., Ataullakhanov F.I., Sinauridze E.I. 2019. Erythrocytes as bioreactors to decrease excess ammonium concentration in blood. Sci. Reports. 9 (1), 1455. CrossRefGoogle Scholar
  12. 12.
    Venediktova N.I., Kosenko E.A., Kaminsky Y.G. 2008. Studies on ammocytes: Development, metabolic characteristics, and detoxication of ammonium. Bull. Exp. Biol. Med. 146, 730–732.CrossRefPubMedGoogle Scholar
  13. 13.
    Kosenko E.A., Venediktova N.I., Kudryavtsev A.A., Ataullakhanov F.I., Kaminsky Y.G., Felipo V., Montoliu C. 2008. Encapsulation of glutamine synthetase in mouse erythrocytes: A new procedure for ammonia detoxification. Biochem. Cell Biol. 86, 469–476.CrossRefPubMedGoogle Scholar
  14. 14.
    Sanz S., Pinilla M., Garin M., Tipton K.F., Luque J. 1995. The influence of enzyme concentration on the encapsulation of glutamate dehydrogenase and alcohol dehydrogenase in red blood cells. Biotechnol. Appl. Biochem. 22, 223–231.PubMedGoogle Scholar
  15. 15.
    Sanz S., Lizano C., Garin M.I., Luque J., Pinilla M. 1997. Biochemical properties of alcohol dehydrogenase and glutamate dehydrogenase encapsulated into human erythrocytes by a hypotonic-dialysis procedure. In: Erythrocytes as drug cariers in medicine. Eds Sprandel U., Way J.L. New York: Springer Science + Business Media, p. 101–108.Google Scholar
  16. 16.
    Millan C.G., Marinero M.L.S., Castaneda A.Z., Lanao J.M. 2004. Drug, enzyme and peptide delivery using erythrocytes as carriers. J. Control. Release. 95, 27–49.CrossRefPubMedGoogle Scholar
  17. 17.
    Gupta A., Mishra A.K., Bansal1 P., Kumar S., Gupta V., Singh R., Kalyan G.S. 2010. Cell based drug delivery system through resealed erythrocyte – a review. Int. J. Pharm. Sci. Drug Res. 2, 23–30.Google Scholar
  18. 18.
    Pierigè F., Serafini S., Rossi L., Magnani M. 2008. Cell-based drug delivery. Adv. Drug Deliv. Rev. 60 (2), 286–295.CrossRefPubMedGoogle Scholar
  19. 19.
    Linderkamp O., Meiselman H.J. 1982. Geometric, osmotic, and membrane mechanical properties of density-separated human red cells. Blood. 59, 1121–1127.CrossRefPubMedGoogle Scholar
  20. 20.
    Ponder E. 1951. Diffractometric measurements of the tonicity volume relations of human red cells in hypotonic systems. J. Gen. Physiol. 34, 567–571.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Evans J., Gratzer W., Mohandas N., Parker K., Sleep J. 2008. Fluctuations of the red blood cell membrane: Relation to mechanical properties and lack of ATP dependence. Biophys. J. 94, 4134–4144.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Seeman P. 1967. Transient holes in the erythrocyte membrane during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin. J. Cell Biol. 32, 55–70.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Saari J.T., Beck J.S. 1975. Hypotonic hemolysis of human red blood cells: A two-phase process. J. Membr. Biol. 23, 212–226.CrossRefGoogle Scholar
  24. 24.
    Erickson H.P. 2009. Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol. Proced Online. 11, 32–51. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Colman R.F. 1990. Dehydrogenases-structure and catalytic mechanisms: Glutamate dehydrogenase (bovine liver). In: A study of enzymes. Ed. S.A. Kuby S.A. Vol. II, Mechanism of enzyme action. Ch. 7. Boston, Roca Raton, Ann Arbor: CRC Press, p. 173–192.Google Scholar
  26. 26.
    Moon K., Smith E.L. 1973. Sequence of bovine liver glutamate dehydrogenase. VIII. Peptides produced by specific chemical cleavages; The complete sequence of the protein. J. Biol. Chem. 248, 3082–3088.PubMedGoogle Scholar
  27. 27.
    Hudson R.C., Daniel R.M. 1993. L-Glutamate dehydrogenases: Distribution, properties and mechanism. Comp. Biochem. Physiol. B. 106, 767–792.CrossRefPubMedGoogle Scholar
  28. 28.
    Goldin B.R., Frieden C. 1971. L-Glutamate dehydrogenases. Curr. Top. Cell. Regul. 4, 77–117.CrossRefGoogle Scholar
  29. 29.
    Sund H., Markau K., Koberstein R. 1975. Glutamate dehydrogenase. In: Subunits in biological systems. Part C (Biological macromolecules. V. 7). Eds Timasheff S.H., Fasman G.D. New York, Basel: Marcel Dekker, p. 225–287.Google Scholar
  30. 30.
    Saier M.H., Jenkins W.T. 1967. Alanine aminotransferase. I. Purification and properties. J. Biol. Chem. 242, 91–100.PubMedGoogle Scholar
  31. 31.
    Eisenberg H., Tomkins G.M. 1968. Molecular weight of the subunits, oligomeric and associated forms of bovine liver glutamate dehydrogenase. J. Mol. Biol. 31, 37–49.CrossRefPubMedGoogle Scholar
  32. 32.
    Sinauridze E.I., Vitvitsky V.M., Pichugin A.V., Zhabotinsky A.M., Ataullakhanov F.I. 1992. A new chemotherapeutic agent: L-Asparaginase entrapped in red blood cells. Adv. Exp. Med. Biol. 326, 203–206.PubMedGoogle Scholar
  33. 33.
    Ihler G.M., Glew R.H., Schnure F.W. 1973. Enzyme loading of erythrocytes. Proc. Natl. Acad. Sci. USA. 70, 2663–2666.CrossRefPubMedGoogle Scholar
  34. 34.
    Godfrin Y., Horand F., Franco R., Dufour E., Kosenko E., Bax B.E., Banz A., Skorokhod O.A., Lanao J.M., Vitvitsky V., Sinauridze E., Bourgeaux V., Gunter K.C. 2012. Meeting highlights: International seminar on the red blood cells as vehicles for drugs. Expert Opin. Biol. Ther. 12, 127–133.CrossRefPubMedGoogle Scholar
  35. 35.
    Yew N.S., Dufour E., Przybylska M., Putelat J., Crawley C., Foster M., Gentry S., Reczek D., Kloss A., Meyzaud A., Horand F., Cheng S.H., Godfrin Y. 2013. Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice. Mol. Genet. Metab. 109, 339–344.CrossRefPubMedGoogle Scholar
  36. 36.
    Santero E., Hervas A.B., Canosa I., Govantes F. 2012. Glutamate dehydrogenases: Enzymology, physiological role and biotechnological relevance. In: Dehydrogenases. Ed. Canuto R.A. InTechOpen. Ch. 12, p. 289–318.
  37. 37.
    McCarthy A.D., Tipton K.F. 1985. Ox glutamate dehydrogenase. Comparison of the kinetic properties of native and proteolysed preparations. Biochem. J. 230, 95–99.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Shcherbachenko I.M., Lisovskaya I.L., Tikhonov V.P. 2007. Oxidation-induced calcium-dependent dehydration of normal human red blood cells. Free Radic. Res. 41, 536–545.CrossRefPubMedGoogle Scholar
  39. 39.
    Klein H.G., Anstee D.J. 2005. Transfusion of blood, blood components and plasma alternatives in oligaemia. In: Mollison’s blood transfusion in clinical medicine. 11 ed. Massachusetts: Blackwell Publishing, Inc., USA. Ch. 2, p. 19–47.CrossRefGoogle Scholar
  40. 40.
    Martinov M.V., Plotnikov A.G., Vitvitsky V.M., Ataullakhanov F.I. 2000. Deficiencies of glycolytic enzymes as a possible cause of hemolytic anemia. Biochim. Biophys. Acta. 1474, 75–87.CrossRefPubMedGoogle Scholar
  41. 41.
    Shimizu H., Kuratsu T., Hirata F. 1979. Purification and some properties of glutamate dehydrogenase from Proteus inconstans. J. Ferment. Technol. 57, 428–433.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2019

Authors and Affiliations

  • D. V. Borsakova
    • 1
    • 2
  • E. S. Protasov
    • 1
    • 2
    • 3
  • S. V. Nazarenko
    • 3
  • Y. G. Alexandrovich
    • 1
    • 2
  • A. A. Butylin
    • 1
    • 3
  • F. I. Ataullakhanov
    • 1
    • 2
    • 3
    • 4
  • E. I. Sinauridze
    • 1
    • 2
    Email author
  1. 1.Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Ministry of HealthcareMoscowRussia
  2. 2.Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of SciencesMoscowRussia
  3. 3.Lomonosov Moscow State University, Faculty of PhysicsMoscowRussia
  4. 4.Moscow Institute of Physics and Technology (National Research University)DolgoprudnyRussia

Personalised recommendations