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Determination of free heme in stored red blood cells with an apo-horseradish peroxidase-based assay

  • Vijith Vijayan , Robert Greite , Sebastian Schott , Julian Doricic , Kukuh Madyaningrana , Pooja Pradhan , Jörg Martens , Rainer Blasczyk , Sabina Janciauskiene und Stephan Immenschuh EMAIL logo
Veröffentlicht/Copyright: 5. September 2022
Biological Chemistry
Aus der Zeitschrift Biological Chemistry Band 403 Heft 11-12

Abstract

Transfusion effectiveness of red blood cells (RBCs) has been associated with duration of the storage period. Storage-dependent RBC alterations lead to hemolysis and release of toxic free heme, but the increase of free heme levels over time is largely unknown. In the current study, an apo-horseradish peroxidase (apoHRP)-based assay was applied to measure levels of free heme at regular intervals or periodically in supernatants of RBCs until a maximum storage period of 42 days. Free heme levels increased with linear time-dependent kinetics up to day 21 and accelerated disproportionally after day 28 until day 42, as determined with the apoHRP assay. Individual time courses of free heme in different RBC units exhibited high variability. Notably, levels of free hemoglobin, an established indicator of RBC damage, and those of total heme increased with continuous time-dependent linear kinetics over the entire 42 day storage period, respectively. Supernatants from RBC units with high levels of free heme led to inflammatory activation of human neutrophils. In conclusion, determining free heme in stored RBCs with the applied apoHRP assay may become feasible for testing of RBC storage quality in clinical transfusion medicine.

Keywords: apoHRP assay; free heme; red blood cells; storage lesion; transfusion
Corresponding author: Stephan Immenschuh, Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany, E-mail:

Funding source: Deutsche Forschungsgemeinschaft

Award Identifier / Grant number: IM 20/4-1

Acknowledgments

We wish to thank Anette Sarti for excellent technical support of the study.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: Supported by grant IM 20/4-1 from the Deutsche Forschungsgemeinschaft, Bonn (Germany) [SI].

  3. Conflict of interest statement: The authors declare no conflicts of interest regarding this article.

References

Adamzik, M., Hamburger, T., Petrat, F., Peters, J., de Groot, H., and Hartmann, M. (2012). Free hemoglobin concentration in severe sepsis: methods of measurement and prediction of outcome. Crit. Care 16: R125, https://doi.org/10.1186/cc11425.Suche in Google Scholar PubMed PubMed Central

Atamna, H., Brahmbhatt, M., Atamna, W., Shanower, G.A., and Dhahbi, J.M. (2015). ApoHRP-based assay to measure intracellular regulatory heme. Metallomics 7: 309–321, https://doi.org/10.1039/c4mt00246f.Suche in Google Scholar PubMed PubMed Central

Baek, J.H., D’Agnillo, F., Vallelian, F., Pereira, C.P., Williams, M.C., Jia, Y., Schaer, D.J., and Buehler, P.W. (2012). Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in Guinea pigs by haptoglobin therapy. J. Clin. Invest. 122: 1444–1458, https://doi.org/10.1172/jci59770.Suche in Google Scholar

Barr, I. and Guo, F. (2015). Pyridine hemochromagen assay for determining the concentration of heme in purified protein solutions. Bio Protoc. 5: e1594, https://doi.org/10.21769/bioprotoc.1594.Suche in Google Scholar PubMed PubMed Central

Belpulsi, D., Spitalnik, S.L., and Hod, E.A. (2017). The controversy over the age of blood: what do the clinical trials really teach us? Blood Transfus 15: 112–115.Suche in Google Scholar

Bennett-Guerrero, E., Veldman, T.H., Doctor, A., Telen, M.J., Ortel, T.L., Reid, T.S., Mulherin, M.A., Zhu, H., Buck, R.D., Califf, R.M., et al.. (2007). Evolution of adverse changes in stored RBCs. Proc. Natl. Acad. Sci. U.S.A 104: 17063–17068, https://doi.org/10.1073/pnas.0708160104.Suche in Google Scholar PubMed PubMed Central

Chasse, M., Tinmouth, A., English, S.W., Acker, J.P., Wilson, K., Knoll, G., Shehata, N., van Walraven, C., Forster, A.J., Ramsay, T., et al.. (2016). Association of blood donor age and sex with recipient survival after red blood cell transfusion. JAMA Intern. Med. 176: 1307–1314, https://doi.org/10.1001/jamainternmed.2016.3324.Suche in Google Scholar PubMed

Chiabrando, D., Vinchi, F., Fiorito, V., Mercurio, S., and Tolosano, E. (2014). Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes. Front. Pharmacol. 5: 61, https://doi.org/10.3389/fphar.2014.00061.Suche in Google Scholar PubMed PubMed Central

Donadee, C., Raat, N.J., Kanias, T., Tejero, J., Lee, J.S., Kelley, E.E., Zhao, X., Liu, C., Reynolds, H., Azarov, I., et al.. (2011). Nitric oxide scavenging by red blood cell microparticles and cell-free hemoglobin as a mechanism for the red cell storage lesion. Circulation 124: 465–476, https://doi.org/10.1161/circulationaha.110.008698.Suche in Google Scholar

Dutra, F.F. and Bozza, M.T. (2014). Heme on innate immunity and inflammation. Front. Pharmacol. 5: 115, https://doi.org/10.3389/fphar.2014.00115.Suche in Google Scholar PubMed PubMed Central

Flegel, W.A., Natanson, C., and Klein, H.G. (2014). Does prolonged storage of red blood cells cause harm? Br. J. Haematol. 165: 3–16, https://doi.org/10.1111/bjh.12747.Suche in Google Scholar PubMed PubMed Central

Francis, R.O., D’Alessandro, A., Eisenberger, A., Soffing, M., Yeh, R., Coronel, E., Sheikh, A., Rapido, F., La Carpia, F., Reisz, J.A., et al.. (2020). Donor glucose-6-phosphate dehydrogenase deficiency decreases blood quality for transfusion. J. Clin. Invest. 130: 2270–2285, https://doi.org/10.1172/jci133530.Suche in Google Scholar PubMed PubMed Central

Gauvin, F., Spinella, P.C., Lacroix, J., Choker, G., Ducruet, T., Karam, O., Hebert, P.C., Hutchison, J.S., Hume, H.A., and Tucci, M. (2010). Association between length of storage of transfused red blood cells and multiple organ dysfunction syndrome in pediatric intensive care patients. Transfusion 50: 1902–1913, https://doi.org/10.1111/j.1537-2995.2010.02661.x.Suche in Google Scholar PubMed

Goel, R., Johnson, D.J., Scott, A.V., Tobian, A.A., Ness, P.M., Nagababu, E., and Frank, S.M. (2016). Red blood cells stored 35 days or more are associated with adverse outcomes in high-risk patients. Transfusion 56: 1690–1698, https://doi.org/10.1111/trf.13559.Suche in Google Scholar PubMed

Graw, J.A., Mayeur, C., Rosales, I., Liu, Y., Sabbisetti, V.S., Riley, F.E., Rechester, O., Malhotra, R., Warren, H.S., Colvin, R.B., et al.. (2016). Haptoglobin or hemopexin therapy prevents acute adverse effects of resuscitation after prolonged storage of red cells. Circulation 134: 945–960, https://doi.org/10.1161/circulationaha.115.019955.Suche in Google Scholar PubMed PubMed Central

Hopp, M.T., Schmalohr, B.F., Kuhl, T., Detzel, M.S., Wissbrock, A., and Imhof, D. (2020). Heme determination and quantification methods and their suitability for practical applications and everyday use. Anal. Chem. 92: 9429–9440, https://doi.org/10.1021/acs.analchem.0c00415.Suche in Google Scholar PubMed

Immenschuh, S., Vijayan, V., Janciauskiene, S., and Gueler, F. (2017). Heme as a target for therapeutic interventions. Front. Pharmacol. 8: 146, https://doi.org/10.3389/fphar.2017.00146.Suche in Google Scholar PubMed PubMed Central

Janciauskiene, S., Tumpara, S., Wiese, M., Wrenger, S., Vijayan, V., Gueler, F., Chen, R., Madyaningrana, K., Mahadeva, R., Welte, T., et al.. (2017). Alpha1-antitrypsin binds hemin and prevents oxidative activation of human neutrophils: putative pathophysiological significance. J. Leukoc. Biol. 102: 1127–1141, https://doi.org/10.1189/jlb.3a0317-124r.Suche in Google Scholar

Kahn, S.E., Watkins, B.F., and Bermes, E.W.Jr. (1981). An evaluation of a spectrophotometric scanning technique for measurement of plasma hemoglobin. Ann. Clin. Lab. Sci. 11: 126–131.Suche in Google Scholar

Kanias, T., Lanteri, M.C., Page, G.P., Guo, Y., Endres, S.M., Stone, M., Keating, S., Mast, A.E., Cable, R.G., Triulzi, D.J., et al.. (2017). Ethnicity, sex, and age are determinants of red blood cell storage and stress hemolysis: results of the REDS-III RBC-Omics study. Blood Adv. 1: 1132–1141, https://doi.org/10.1182/bloodadvances.2017004820.Suche in Google Scholar PubMed PubMed Central

Kim-Shapiro, D.B., Lee, J., and Gladwin, M.T. (2011). Storage lesion: role of red blood cell breakdown. Transfusion 51: 844–851, https://doi.org/10.1111/j.1537-2995.2011.03100.x.Suche in Google Scholar PubMed PubMed Central

Kumar, S. and Bandyopadhyay, U. (2005). Free heme toxicity and its detoxification systems in human. Toxicol. Lett. 157: 175–188, https://doi.org/10.1016/j.toxlet.2005.03.004.Suche in Google Scholar PubMed

Larsen, R., Gozzelino, R., Jeney, V., Tokaji, L., Bozza, F.A., Japiassu, A.M., Bonaparte, D., Cavalcante, M.M., Chora, A., Ferreira, A., et al.. (2010). A central role for free heme in the pathogenesis of severe sepsis. Sci. Transl. Med. 2: 51ra71, https://doi.org/10.1126/scitranslmed.3001118.Suche in Google Scholar PubMed

Lee, J.S. and Kim-Shapiro, D.B. (2017). Stored blood: how old is too old? J. Clin. Invest. 127: 100–102, https://doi.org/10.1172/jci91309.Suche in Google Scholar

Muller Eberhard, U. (1970). Hemopexin. N. Engl. J. Med. 283: 1090–1094, https://doi.org/10.1056/nejm197011122832007.Suche in Google Scholar

Oh, J.Y., Hamm, J., Xu, X., Genschmer, K., Zhong, M., Lebensburger, J., Marques, M.B., Kerby, J.D., Pittet, J.F., Gaggar, A., et al.. (2016). Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices. Redox Biol. 9: 167–177, https://doi.org/10.1016/j.redox.2016.08.003.Suche in Google Scholar PubMed PubMed Central

Rapido, F., Brittenham, G.M., Bandyopadhyay, S., La Carpia, F., L’Acqua, C., McMahon, D.J., Rebbaa, A., Wojczyk, B.S., Netterwald, J., Wang, H., et al.. (2017). Prolonged red cell storage before transfusion increases extravascular hemolysis. J. Clin. Invest. 127: 375–382, https://doi.org/10.1172/jci90837.Suche in Google Scholar

Roubinian, N.H., Reese, S.E., Qiao, H., Plimier, C., Fang, F., Page, G.P., Cable, R.G., Custer, B., Gladwin, M.T., Goel, R., et al.. (2022). Donor genetic and nongenetic factors affecting red blood cell transfusion effectiveness. JCI Insight. 7: e152598, https://doi.org/10.1172/jci.insight.152598.Suche in Google Scholar PubMed PubMed Central

Schaer, D.J., Buehler, P.W., Alayash, A.I., Belcher, J.D., and Vercellotti, G.M. (2012). Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood 121: 1276–1284, https://doi.org/10.1182/blood-2012-11-451229.Suche in Google Scholar PubMed PubMed Central

Semple, E., Bowes-Schmidt, A., Yi, Q.L., Shimla, S., and Devine, D.V. (2012). Transfusion reactions: a comparative observational study of blood components produced before and after implementation of semiautomated production from whole blood. Transfusion 52: 2683–2691, https://doi.org/10.1111/j.1537-2995.2012.03752.x.Suche in Google Scholar PubMed

Soares, M.P. and Bozza, M.T. (2016). Red alert: labile heme is an alarmin. Curr. Opin. Immunol. 38: 94–100, https://doi.org/10.1016/j.coi.2015.11.006.Suche in Google Scholar PubMed

Stapley, R., Rodriguez, C., Oh, J.Y., Honavar, J., Brandon, A., Wagener, B.M., Marques, M.B., Weinberg, J.A., Kerby, J.D., Pittet, J.F., et al.. (2015). Red blood cell washing, nitrite therapy, and antiheme therapies prevent stored red blood cell toxicity after trauma-hemorrhage. Free Radic. Biol. Med. 85: 207–218, https://doi.org/10.1016/j.freeradbiomed.2015.04.025.Suche in Google Scholar PubMed PubMed Central

Sudan, K., Vijayan, V., Madyaningrana, K., Gueler, F., Igarashi, K., Foresti, R., Motterlini, R., and Immenschuh, S. (2019). TLR4 activation alters labile heme levels to regulate BACH1 and heme oxygenase-1 expression in macrophages. Free Radic. Biol. Med. 137: 131–142, https://doi.org/10.1016/j.freeradbiomed.2019.04.024.Suche in Google Scholar PubMed

Taketani, S., Immenschuh, S., Go, S., Sinclair, P.R., Stockert, R.J., Liem, H.H., and Muller Eberhard, U. (1998). Hemopexin from four species inhibits the association of heme with cultured hepatoma cells or primary rat hepatocytes exhibiting a small number of species specific hemopexin receptors. Hepatology 27: 808–814, https://doi.org/10.1002/hep.510270324.Suche in Google Scholar PubMed

Tinmouth, A., Fergusson, D., Yee, I.C., and Hebert, P.C. (2006). Clinical consequences of red cell storage in the critically ill. Transfusion 46: 2014–2027, https://doi.org/10.1111/j.1537-2995.2006.01026.x.Suche in Google Scholar PubMed

Wagener, B.M., Hu, P.J., Oh, J.Y., Evans, C.A., Richter, J.R., Honavar, J., Brandon, A.P., Creighton, J., Stephens, S.W., Morgan, C., et al.. (2018). Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: a preclinical experimental study. PLoS Med. 15: e1002522, https://doi.org/10.1371/journal.pmed.1002522.Suche in Google Scholar PubMed PubMed Central

Wang, L., Vijayan, V., Jang, M.S., Thorenz, A., Greite, R., Rong, S., Chen, R., Shushakova, N., Tudorache, I., Derlin, K., et al.. (2019). Labile heme aggravates renal inflammation and complement activation after ischemia reperfusion injury. Front. Immunol. 10: 2975, https://doi.org/10.3389/fimmu.2019.02975.Suche in Google Scholar PubMed PubMed Central

Yuan, X., Rietzschel, N., Kwon, H., Walter Nuno, A.B., Hanna, D.A., Phillips, J.D., Raven, E.L., Reddi, A.R., and Hamza, I. (2016). Regulation of intracellular heme trafficking revealed by subcellular reporters. Proc. Natl. Acad. Sci. U.S.A 113: E5144–E5152, https://doi.org/10.1073/pnas.1609865113.Suche in Google Scholar PubMed PubMed Central

Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2022-0184).

Received: 2022-05-17
Revised: 2022-08-10
Accepted: 2022-08-15
Published Online: 2022-09-05
Published in Print: 2022-11-25

© 2022 Walter de Gruyter GmbH, Berlin/Boston

Artikel in diesem Heft

  1. Frontmatter
  2. Heme research – the past, the present and the future
  3. A primer on heme biosynthesis
  4. New roles for GAPDH, Hsp90, and NO in regulating heme allocation and hemeprotein function in mammals
  5. The role of host heme in bacterial infection
  6. Signal transduction mechanisms in heme-based globin-coupled oxygen sensors with a focus on a histidine kinase (AfGcHK) and a diguanylate cyclase (YddV or EcDosC)
  7. Heme delivery to heme oxygenase-2 involves glyceraldehyde-3-phosphate dehydrogenase
  8. Novel insights into heme binding to hemoglobin
  9. Extracellular hemin is a reverse use-dependent gating modifier of cardiac voltage-gated Na+ channels
  10. Assessment of the breadth of binding promiscuity of heme towards human proteins
  11. Determination of free heme in stored red blood cells with an apo-horseradish peroxidase-based assay
  12. Comparative studies of soluble and immobilized Fe(III) heme-peptide complexes as alternative heterogeneous biocatalysts
https://doi.org/10.1515/hsz-2022-0184
Schlagwörter für diesen Artikel
apoHRP assay; free heme; red blood cells; storage lesion; transfusion

Artikel in diesem Heft

  1. Frontmatter
  2. Heme research – the past, the present and the future
  3. A primer on heme biosynthesis
  4. New roles for GAPDH, Hsp90, and NO in regulating heme allocation and hemeprotein function in mammals
  5. The role of host heme in bacterial infection
  6. Signal transduction mechanisms in heme-based globin-coupled oxygen sensors with a focus on a histidine kinase (AfGcHK) and a diguanylate cyclase (YddV or EcDosC)
  7. Heme delivery to heme oxygenase-2 involves glyceraldehyde-3-phosphate dehydrogenase
  8. Novel insights into heme binding to hemoglobin
  9. Extracellular hemin is a reverse use-dependent gating modifier of cardiac voltage-gated Na+ channels
  10. Assessment of the breadth of binding promiscuity of heme towards human proteins
  11. Determination of free heme in stored red blood cells with an apo-horseradish peroxidase-based assay
  12. Comparative studies of soluble and immobilized Fe(III) heme-peptide complexes as alternative heterogeneous biocatalysts
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