Enzyme protection systems of erythrocytes in conditions of ascorbate recirculation and oxidative loading

Keywords: oxidative stress; glutathione; transmembrane transport of electrons; copper ions; chelation complexes of o-phenanthroline


Vitamin C was shown to partially protect red blood cells from oxidative changes during storage by noticeable reduction of mechanical fragility and hemolysis. In order to maintain the content of ascorbate in the reconstituted form in plasma, the latter is involved in a number of oxidative-reducing processes within red blood cells. This work is a continuation of studies of the effects of ascorbate on the metabolic processes that maintain the viability of red blood cells. Human red blood cells were incubated for five hours at 25 ºC in the oxidizing media system 1 1.0 · 10–4 M ascorbic acid (AscH), 5 · 10–6 M Cu2+, Na-phosphate buffer (0.015 M, pH 7.4), 0.15 M NaCl, and system 2, that contained o-phenanthroline at a concentration of 1.0 · 10–4 M in addition to the components of system 1 medium. For these cells, the changes in the content of reduced glutathione, glutathione enzyme activity, and the state of the membrane electron transport NADH: ferricyanide reductase were determined in time. The obtained data indicate that red blood cells undergo significant oxidative stress under the influence of the oxidative medium. During the first incubation period of erythrocytes in the AscH-Cu2+ environment, the activity of glutathione peroxidase and glutathione-S-transferase reached the maximum values, indicating the presence of H2O2 in the cell and the activation of lipid peroxidation processes. Glutathione-S-transferase activity remained above the control level throughout the entire study period. The activity of glutathione reductase and glucose-6-phosphate dehydrogenase was reduced. The oxidative loading of erythrocytes in the presence of o-phenanthroline was lower, the development of oxidative stress occurred in 90 minutes, but the binding of the o-phenanthroline complexes of Cu2+ to the membrane modified the SH-group of membrane proteins and this reduced the transport capabilities of the dehydroascorbate transporters and the electron transmembrane system, the consequence of which may be the accumulation of oxidized forms of ascorbate outside. We detected the participation of CO-signaling mechanism in hemoglobin deglutathionylation and increase in the content of glutathione. In this work we discuss the role of metabolic reprogramming in red blood cells through thiol-disulfide exchange as a mechanism that can be involved into adaptive responses aimed at counteracting stress in mammalian tissues.


Arese, P., Gallo, V., Pantaleo, A., & Turrini, F. (2012). Life and death of glucose-6-phosphate dehydrogenase (G6PD) deficient erythrocytes role of redox stress and band 3 modifications. Transfusion Medicine and Hemotherapy, 39(5), 328–334.

Dotsenko, O. I., Dragushenko, O. O., & Dotsenko, V. A. (2010). The investigation of the action of prooxidant and cytotoxic systems Cu2+–AscH, Cu2+–AscH–o-phenanthroline [Doslidzhennia prooksydantnoi ta tsytotoksychnoi dii system Cu2+–AscH, Cu2+–AscH–o-phenanthroline]. Dosiahnennia Biolohii ta Medytsyny, 15(1), 1–7 (in Ukrainian).

Dotsenko, O. I. (2015). Glutathione system’s activity in the blood of mice in the conditions of vibration stress [Aktivnost' sistemy glutationa krovi myshej, nahodjashhihsja v uslovijah vibracionnogo stressa]. ScienceRise, 11(16), 39–46 (in Russian).

Carelli-Alinovi, C., & Misiti, F. (2017). Erythrocytes as potential link between diabetes and Alzheimer's disease. Frontiers in Aging Neuroscience, 25(9), 276–286.

Chikezie, C. P. (2011). Glutathione S-transferase activity of human erythrocytes incubated in aqueous solutions of five antimalarial drugs. Free Radicals and Antioxidants, 1(2), 26–30.

Corti, A., Casini, A. F., & Pompella, A. (2010). Cellular pathways for transport and efflux of ascorbate and dehydroascorbate. Archives of Biochemistry and Biophysics, 500, 107–115.

Giustarini, D., Colombo, G., Garavaglia, M. L., Astori, E., Portinaro, N. M., Reggiani, F., Badalamenti, S., Aloisi, A. M., Santucci, A., Rossi, R., Milzani, A., & Dalle-Donne, I. (2017). Assessment of glutathione/glutathione disulphide ratio and S-glutathionylated proteins in human blood, solid tissues, and cultured cells. Free Radical Biology and Medicine, 112, 360–375.

Crane, F. L., Crane, H. E., Sun, I. L., MacKellar, W. C., Grebing, C., & Löw, H. (1982). Insulin control of a transplasma membrane NADH dehydrogenase in erythrocyte membranes. Journal of Bioenergetics and Biomembranes, 14(5–6), 425–433.

Hiroshige, Y. (1980). The effects of copper and copper-o-phenanthroline complex on the intact human erythrocytes. The Tohoku Journal of Experimental Medicine, 130, 385–402.

Kennett, E. C., & Kuchel, P. W. (2006). Plasma membrane oxidoreductases: Effects on erythrocyte metabolism and redox homeostasis. Antioxidants and Redox Signaling, 8(7–8), 1241–1247.

Kuhn, V., Diederich, L., Keller, T. C. S. IV, Kramer, C. M., Lückstädt, W., Panknin, C., Suvorava, T., Isakson, B. E., Kelm, M., & Cortese-Krott, M. M. (2017). Red blood cell function and dysfunction: Redox regulation, nitric oxide metabolism, anemia. Antioxidants and Redox Signaling, 26(13), 718–742.

Li, H., Tu, H., Wang, Y., & Levine, M. (2012). Vitamin C in mouse and human red blood cells: An HPLC assay. Analytical Biochemistry, 426(2), 109–117.

Lu, Y. X., Wu, Q. N., Chen, D. L., Chen, L. Z., Wang, Z. X., Ren, C., Mo, H. Y., Chen, Y., Sheng, H., Wang, Y. N., Wang, Y., Lu, J. H., Wang, D. S., Zeng, Z. L., Wang, F., Wang, F. H., Li, Y. H., Ju, H. Q., & Xu, R. H. (2018). Pharmacological ascorbate suppresses growth of gastric cancer cells with GLUT1. Overexpression and enhances the efficacy of oxaliplatin through redox modulation. Theranostics, 8(5), 1312–1326.

Mannervik, B. (2001). Measurement of glutathione reductase activity. Current Protocols in Toxicology, 7, 7.2.

Matteucci, E., & Giampietro, O. (2007). Electron pathways through erythrocyte plasma membrane in human physiology and pathology: Potential redox biomarker? Biomarker Insights, 2, 321–329.

Maurya, P. K., Kumar, P., & Chandra, P. (2015). Biomarkers of oxidative stress in erythrocytes as a function of human age. World Journal of Methodology, 5(4), 216–222.

May, J. M., Qu, Z.-C., & Morrow, J. D. (1996). Interaction of ascorbate and α-tocopherol in resealed human erythrocyte ghosts: Transmembrane electron transfer and protection from lipid peroxidation. The Journal of Biological Chemistry, 271, 10577–10582.

May, J. M., Qu, Z. C., & Cobb, C. E. (2004). Human erythrocyte recycling of ascorbic acid: Relative contributions from the ascorbate free radical and dehydroascorbic acid. Journal of Biological Chemistry, 279(15), 14975–14982.

Metere, A., Iorio, E., Scorza, G., Camerini, S., Casella, M., Crescenzi, M, Minetti, M., & Pietraforte, D. (2014). Carbon monoxide signaling in human red blood cells: Evidence for pentose phosphate pathway activation and protein deglutathionylation. Antioxidants and Redox Signaling, 20(3), 403–416.

O'Leary, B. R., Houwen, F. K., Johnson, C. L., Allen, B. G., Mezhir, J. J., Berg, D. J., Cullen, J. J., & Spitz, D. R. (2018). Pharmacological ascorbate as an adjuvant for enhancing radiation-chemotherapy responses in gastric adenocarcinoma. Radiation Research, 189(5), 456–465.

Ou, P., & Wolff, S. P. (1996). A discontinuous method for catalase determination at 'near physiological' concentrations of H2O2 and its application to the study of H2O2 fluxes within cells. Journal of Biochemical and Biophysical Methods, 31(1–2), 59–67.

Pandey, K. B., & Rizvi, S. I. (2010). Markers of oxidative stress in erythrocytes and plasma during aging in humans. Oxidative Medicine and Cellular Longevity, 3(1), 2–12.

Padayatty, S. J., & Levine, M. (2016). Vitamin C: The known and the unknown and goldilocks. Oral Diseases, 22(6), 463–493.

Razygrayev, A. V., & Arutyunyan, A. V. (2006). Determination of human serum glutathione peroxidase activity, by using hydrogen peroxide and 5,5’-dithio-bis (2-nitrobenzoic acid) [Opredelenie glutationperoksidaznoj aktivnosti v syvorotke krovi cheloveka s ispol'zovaniem peroksida vodoroda i 5,5’-ditiobis(2-nitrobenzojnoj kisloty)]. Klinicheskaia Laboratornaia Diagnostika, 6, 13–16 (in Russian).

Reisz, J. A., Wither, M. J., Dzieciatkowska, M., Nemkov, T., Issaian, A., Yoshida, T., Dunham, A. J., Hill, R. C., Hansen, K. C., & D'Alessandro, A. (2016). Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells. Blood, 128(12), 32–42.

Rinalducci, S., Marrocco, C., & Zolla, L. (2015) Thiol-based regulation of glyceraldehyde-3-phosphate dehydrogenase in blood bank-stored red blood cells: A strategy to counteract oxidative stress. Transfusion, 55(3), 499–506.

Sanford, K., Fisher, B. J., Fowler, E., Fowler, A. A., & Natarajan, R. (2017). Attenuation of red blood cell storage lesions with vitamin C. Antioxidants (Basel), 6(3), e55.

Shan, G., Yang, F., Zhou, L., Tang, T., Okoro, E. U., Yang, H., & Guo, Z. (2015). Increase in blood glutathione and erythrocyte proteins related to glutathione generation, reduction and utilization in African-American old women with diabetes. Journal of Environmental Science and Technology, 5(1), 3000251.

Scarpa, M. (1996). Ascorbate oxidation catalyzed by bis(histidine) copper (II). Inorganic Chemistry, 35(18), 5201–5206.

Soumya, R., & Vani, R. (2017). Vitamin C as a modulator of oxidative stress in erythrocytes of stored blood. Acta Haematologica Polonica, 48(4), 350–356.

Svistunenko, D. A., Dunne, J., Fryer, M., Nicholls, P., Reeder, B. J., Wilson, M. T., Bigotti, M. G., Cutruzzolà, F., & Cooper, C. E. (2002) Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide. Biophysical Journal, 83(5), 2845–2855.

Su, D., May, J. M., Koury, M. J., & Asard, H. (2006). Human erythrocyte membranes contain a cytochrome b561 that may be involved in extracellular ascorbate recycling. Journal of Biological Chemistry, 281, 39852–39859.

Tousova, K., Susankova, K., Teisinger, J., Vyklicky, L., & Vlachova, V. (2004). Oxidizing reagent copper-o-phenanthroline is an open channel blocker of the vanilloid receptor TRPV1. Neuropharmacology, 47(2), 273–285.

Tu, H., Li, H., Wang, Y., Niyyati, M., Wang, Y., Leshin, J., & Levine, M. (2015). Low red blood cell vitamin C concentrations induce red blood cell fragility: A link to diabetes via glucose, glucose transporters, and dehydroascorbic acid. EBioMedicine, 2(11), 1735–1750.

Tu, H., Wang, Y., Li, H., Brinster, L. R., & Levine, M. (2017). Chemical transport knockout for oxidized vitamin C, dehydroascorbic acid, reveals its functions in vivo. EBioMedicine, 23, 125–135.

VanDuijn, M. M., Tijssen, K., VanSteveninck, J., Van Den Broek, P. J., & Van Der Zee, J. (2000). Erythrocytes reduce extracellular ascorbate free radicals using intracellular ascorbate as an electron donor. Journal of Biological Chemistry, 275, 27720–27725.

Vani, R., Soumya, R., Carl, H., Chandni, V. A., Neha, K., Pankhuri, B., Trishna, S., & Vatsal, D. P. (2015). Prospects of vitamin C as an additive in plasma of stored blood. Advances in Hematology, 4, 961049.

Witmer, J. R., Wetherell, B. J., Wagner, B. A., Du, J., Cullen, J. J., & Buettner, G. R. (2016). Direct spectrophotometric measurement of supra-physiological levels of ascorbate in plasma. Redox Biology, 8, 298–304.

Xu, D. P., Washburn, M. P., Sun, G. P., & Wells, W. W. (1996). Purification and characterization of a glutathione dependent dehydroascorbate reductase from human erythrocytes. Biochemical and Biophysical Research Communications, 221(1), 117–121.

Xu, J., & Jordan, R. B. (1990). Kinetics and mechanism of the reaction of aqueous copper (II) with ascorbic acid. Inorganic Chemistry, 29(16), 2933–2936.

Zhang, Z. Z., Lee, E. E., Sudderth, J., Yue, Y., Zia, A., Glass, D., Deberardinis, R. J., & Wang, R. C. (2016). Glutathione depletion, pentose phosphate pathway activation, and hemolysis in erythrocytes protecting cancer cells from vitamin C-induced oxidative Stress. Journal of Biological Chemistry, 291(44), 22861–22867.

How to Cite
Dotsenko, O. I., Taradina, G. V., & Voronych, M. V. (2018). Enzyme protection systems of erythrocytes in conditions of ascorbate recirculation and oxidative loading. Regulatory Mechanisms in Biosystems, 9(4), 584-590. https://doi.org/10.15421/021887