Energy Production and Redox Status of Rat Red Blood Cells after Reticulocytosis Induced by Various Treatments


Stimulated erythropoiesis and reticulocytosis can be induced by daily bleeding, or by phenylhydrazine (PHZ) treatment. We compared the in vivo effects of PHZ and bleeding treatment on haematological, energy and redox status parameters in red blood cells (RBC) of rats. The results showed that all followed haematological parameters were significantly lower in bleeding, compared to PHZ-treated rats. PHZ induced even 2.58-fold higher reticulocytosis as compared to bleeding treatment. Although PHZ induced higher reticulocytosis, respiration intensity and energy production was lower than in bleeding-induced reticulocytes. These alterations were the consequence of increased superoxide anion and peroxynitrite concentrations in PHZ-treated rats. Bleeding treatment resulted in increased activity of an antioxidative enzyme, superoxide dismutase. In conclusion, differences in these two experimental models for reticulocytosis may be used as tools for appropriate pharmacological testing of redox-active substances considering energy and redox processes, as well as apoptosis pathways.


  1. 1.

    Auclair, C., Voisin, E. (1985) Nitroblue tetrazolium reduction. In: Greenwald, R. A. (ed.) Handbook of Methods for Oxygen Radical Research. CRC Press, Inc., Boca Raton, pp. 123–132.

    Google Scholar 

  2. 2.

    Bates, D. A., Winterbourn, C. C. (1984) Haemoglobin denaturation, lipid peroxidation and haemolysis in phenylhydrazine-induced anemia. Biochem. Biophys. Acta 798, 84–87.

    CAS  Article  Google Scholar 

  3. 3.

    Beutler, E. (1982) Catalase. In: Beutler, E. (ed.) Red Cell Metabolism. A Manual of Biochemical Methods. Grune and Stratton, Inc., New York, pp. 105–106.

    Google Scholar 

  4. 4.

    Brown, G. C., Borutaite, V. (2002) Nitric oxide inhibition of mitochondrial respiration and its role in cell death. Free Radic. Biol. Med. 33, 1440–1450.

    CAS  Article  Google Scholar 

  5. 5.

    Diwan, A., Koesters, A. G., Capella, D., Geiger, H., Kalfa, T. A., Dorn, G. W. 2nd (2008) Targeting erythroblast-specific apoptosis in experimental anemia. Apoptosis 13, 1022–1030.

    Article  Google Scholar 

  6. 6.

    Drabkin, D., Austin, H. (1935) Spectrophotometric studies preparations from washed blood cells. J. Biol. Chem. 112, 51–55.

    CAS  Google Scholar 

  7. 7.

    Freedman, M.L., Karpatkin, S. (1975) Heterogeneity of rabbit platelets. IV. Thrombocytosis with absolute megathrombocytosis in phenylhydrazine-induced hemolytic anemia in rabbits. Thromb. Diath. Haemorrh. 33, 335–340.

    CAS  Article  Google Scholar 

  8. 8.

    Gonchar, O., Mankovskaya, I. (2009) Effect of moderate hypoxia/reoxygenation on mitochondrial adaptation to acute severe hypoxia. Acta Biol. Hung. 60, 185–194.

    Article  Google Scholar 

  9. 9.

    Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., Tannenbaum, S. R. (1982) Analysis of nitrate, nitrite and [15N]nitrate in biological fluids. Anal. Biochem. 126, 131–138.

    CAS  Article  Google Scholar 

  10. 10.

    Kostić, M. M., Živković, R. V., Rapoport, S. M. (1990) Maturation-dependent changes of the rat reticulocyte energy metabolism and hormonal responsiveness. Biomed. Biochim. Acta 49, 178–182.

    Google Scholar 

  11. 11.

    Magnani, M., Rossi, L., Cucchiarini, L., Stocchi, V., Fornanini, G. (1988) Effect of phenylhydrazine on red blood cell metabolism. Cell Biochem. Funct. 6, 175–182.

    CAS  Article  Google Scholar 

  12. 12.

    Maletić, S. D., Kostić, M. M. (1999) Effects of nitroglycerin on energy metabolism of rat reticulocytes. J. Physiol. Pharmacol. 50, 75–87.

    PubMed  Google Scholar 

  13. 13.

    Marklund, S., Marklund, G. (1974) Involvement of superoxide anion radical in the autoxidation of pyrogallol and convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474.

    CAS  Article  Google Scholar 

  14. 14.

    Marković, S. D., Ognjanović, B. I., Štajn, A. Š., Žikić, R. V., Saičić, Z. S., Radojičić, R. M., Spasić, M. B. (2006) The effects of nitroglycerine on the redox status of rat erythrocytes and reticulocytes. Physiol. Res. 55, 389–396.

    PubMed  Google Scholar 

  15. 15.

    Marković, S. D., Vukajlović, M. Dj., Ognjanović, B. I., Štajn, A. Š., Žikić, R. V., Saičić, Z. S., Radojičić, R. M., Jones, D. R., Spasić, M. B. (2007) The effects of molsidomine and 3-morpholinosydnonimine on redox status of rat erythrocytes and reticulocytes: a comparative study. Cell Biochem. Funct. 25, 251–258.

    Article  Google Scholar 

  16. 16.

    Marković, S. D., Milošević, M. P., Djordjević, N. Z., Ognjanović, B. I., Štajn, A. Š., Saičić, Z. S., Spasić, M. B. (2009) Time course of hematological parameters in bleeding-induced anemia. Arch. Biol. Sci. 61, 165–170.

    Article  Google Scholar 

  17. 17.

    McMillan, D. C., Powell, C. L., Bowman, Z. S., Morrow, J. D., Jollow, D. J. (2005) Lipids versus proteins as major targets of pro-oxidant, direct-acting hemolytic agents. Toxicol. Sci. 88, 274–283.

    CAS  Article  Google Scholar 

  18. 18.

    Nakanishi, A., Kinuta, K., Abe, T., Araki, K., Yoshida, Y., Liang, S., Li, A., Takei, K., Kinuta, M. (2003) Formation of meso, N-diphenylprotoporphyrin IX by an aerobic reaction of phenylhydrazine with oxyhemoglobins. Acta Med. Okayama 57, 249–256.

    CAS  PubMed  Google Scholar 

  19. 19.

    Pick, E., Keisari, Y. (1980) A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. Immunol. Meth. 38, 161–170.

    CAS  Article  Google Scholar 

  20. 20.

    Ramot, Y., Koshkaryev, A., Goldfarb, A., Yedgar, S., Barshtein, G. (2008) Phenylhydrazine as a partial model for β-thalassemia red blood cell hemodynamic properties. Br. J. Haematol. 140, 692–700.

    CAS  Article  Google Scholar 

  21. 21.

    Rapoport, S. M. (1986) The Reticulocyte. CRC Press, Inc., Boca Raton, Florida.

    Google Scholar 

  22. 22.

    Redondo, P. A., Alvarez, A. I., Diez, C., Fernandez-Rojo, F., Prieto, J. G. (1995) Physiological response to experimentally induced anemia in rats: a comparative study. Lab. Anim. Sci. 45, 578–583.

    CAS  PubMed  Google Scholar 

  23. 23.

    Riordan, J. F., Vallee, B. L. (1972) Nitration with tetranitromethane. In: Hirs, C. H. W., Timasheff, S. N. (eds) Methods in Enzymology. Academic Press, New York, pp. 515–521.

    Google Scholar 

  24. 24.

    Savill, N. J., Chadwick, W., Reece, S. E. (2009) Quantitative analysis of mechanisms that govern red blood cell age structure and dynamics during anaemia. PLOS Comput. Biol. 5, 1–19.

    Article  Google Scholar 

  25. 25.

    Siems, W., Müller, M., Dumdey, R., Holzhütter, H. G., Rathmann, J., Rapoport, S. M. (1982) Quantification of pathways of glucose utilization and balance of energy metabolism of rabbit reticulocytes. Eur. J. Biochem. 124, 567–576.

    CAS  Article  Google Scholar 

  26. 26.

    Stern, A. (1989) Drug-induced oxidative denaturation in red blood cells. Semin. Hematol. 26, 301–306.

    CAS  PubMed  Google Scholar 

  27. 27.

    Umbreit, W. W., Burris, R. H., Stauffer, F. (1964) Manometric Techniques. Burgess Publishing Co., Minneapolis, pp. 1–17.

    Google Scholar 

  28. 28.

    Živković, R. V., Kostić, M. M., Siems, W., Werner, A., Mojsilović, L. P., Gerber, G. (1990) Effects of phenylhydrazine hydrochloride on energy metabolism in rabbit erythrocytes and reticulocytes. Biomed. Biochim. Acta 49, 172–177.

    Google Scholar 

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Correspondence to Snežana D. Marković.

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Marković, S.D., Žižić, J.B., Obradović, A.D. et al. Energy Production and Redox Status of Rat Red Blood Cells after Reticulocytosis Induced by Various Treatments. BIOLOGIA FUTURA 62, 122–132 (2011).

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  • Bleeding
  • energy production
  • phenylhydrazine
  • redox status
  • reticulocytes