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Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes

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Abstract

Unconjugated bilirubin increasingly binds to erythrocytes as the bilirubin-to-albumin molar ratio exceeds unity, leading to toxic manifestations that can culminate in cell lysis. Our previous studies showed that bilirubin induces the release of lipids from erythrocyte membranes. In the present work, those studies were extended in order to characterize the alterations of membrane lipid composition and evaluate whether bilirubin leads to a loss of phospholipid asymmetry. To this end, human erythrocytes were incubated with several bilirubin-to-albumin molar ratios (0.5 to 5), and cholesterol as well as the total and the individual classes of phospholipids were determined. To detect erythrocytes with phosphatidylserine at the outer surface, the number of annexin V-positive cells was determined following incubation with bilirubin, fixing its molar ratio to albumin at 3. The results demonstrate profound changes in erythrocyte membrane composition, including modified cholesterol and phospholipid content. The release of membrane cholesterol, as well as of total and individual classes of phospholipids at molar ratios ≥1, indicates that damage of erythrocytes may occur in severely ill jaundiced neonates. The loss of the inner-located phospholipids, phosphatidylethanolamine and phosphatidylserine, points to a redistribution of phospholipids in the membrane bilayer. This was confirmed by the exposure of phosphatidylserine at the outer cell surface. In conclusion, this study demonstrates that bilirubin induces loss of membrane lipids and externalization of phosphatidylserine in human erythrocytes. These features may facilitate hemolysis and erythrophagocytosis, thus contributing to enhanced bilirubin production and anemia during severe neonatal hyperbilirubinemia.

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References

  • Ahifors CE. Criteria for exchange transfusion in jaundiced newborns. Pediatrics. 1994:93:488–94.

    Google Scholar 

  • Baldwin JM, O'Reilly R, Whitney M, Lucy JA. Surface exposure of phosphatidylserine is associated with the swelling and osmotically-induced fusion of human erythrocytes in the presence Of a2+. Biochim Biophys Acta. 1990:1028: 14–20.

    Article  PubMed  CAS  Google Scholar 

  • Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem. 1959:234:466–8.

    PubMed  CAS  Google Scholar 

  • Belanger S, Lavoie JC, Chessex P. Influence of bilirubin on the antioxidant capacity of plasma in newborn infants. Biol Neonate. 1997:71:233–8.

    PubMed  CAS  Google Scholar 

  • Boas FE, Forman L, Beutler E. Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia. Proc Nati Acad Sci USA. 1998:95:3077–81.

    Article  CAS  Google Scholar 

  • Bonillo-Perales A, Munoz-Hoyos A, Martinez-Morales A, Molina-Carballo A, Uberos-Fernandez J, Puertas-Prieto A. Changes in erythrocytic deformability and plasma visc-osity in neonatal ictericia. Am J Perinatol. 1999:16:421–7.

    Article  PubMed  CAS  Google Scholar 

  • Bonomini M, Sirolli V, Settefrati N, Dottori S, Di Liberate L, Arduini A. Increased erythrocyte phosphatidylserine expo-sure in chronic renal failure. J Am Soc Nephrol. 1999:10: 1982–90.

    PubMed  CAS  Google Scholar 

  • Bratlid D. Bilirubin binding by human erythrocytes. Scand J Clin Lab Invest. 1972:29:91–7.

    PubMed  CAS  Google Scholar 

  • Brites D, Silva R, Brito A. Effect of bilirubin on erythrocyte shape and haemolysis, under hypotonic, aggregating or non-aggregating conditions, and correlation with cell age. Scand J Clin Lab Invest. 1997:57:337–50.

    Article  PubMed  CAS  Google Scholar 

  • Brito MA. InteracCBo da bilirrubina corn o eritrocito. Especies moleculares envolvidas, estadios de toxicidade e comporta-mento da celula fetal. PhD thesis. University of Lisbon; 2001.

  • Brito MA, Silva RM, Matos DC, da Silva AT, Brites DT. Alterations of erythrocyte morphology and lipid composi-tion by hyperbilirubinemia. Clin Chim Acta. 1996:249:149–65.

    Article  PubMed  CAS  Google Scholar 

  • Brito MA, Silva R, Tiribelli C, Brites D. Assessment of bilirubin toxicity to erythrocytes. Implication in neonatal jaundice management. Eur J Clin Invest. 2000:30:239–47.

    Article  PubMed  CAS  Google Scholar 

  • Brito MA, Brondino CD, Moura JJG, Brites D. Effects of bilirubin molecular species on membrane dynamic proper-ties of human erythrocyte membranes: A spin label electron paramagnetic resonance spectroscopy study. Arch Biochem Biophys. 2001:387:57–65.

    Article  PubMed  CAS  Google Scholar 

  • Brodersen R. Binding of bilirubin to albumin. CRC Crit Rev ClinLabSci. 1980:11:305–401.

    CAS  Google Scholar 

  • Brodersen R, Stern L. Deposition of bilirubin acid in the central nervous system-a hypothesis for the development ofkernicterus. Acta Paediatr Scand. 1990:79:12–9.

    PubMed  CAS  Google Scholar 

  • Butikofer P, Lin ZW, Chiu DT, Lubin B, Kuypers FA. Transbi-layer distribution and mobility ofphosphoinositol in human red blood cells. J Biol Chem. 1990:265:16035–8.

    PubMed  CAS  Google Scholar 

  • Cashore WJ. Free bilirubin concentrations and bilirubin-bind-ing affinity in term and preterm infants. J Pediatr. 1980:96: 521–7.

    Article  PubMed  CAS  Google Scholar 

  • Cheung WH, Sawitsky A, lsenberg HD. The effect of bilirubin on the mammalian erythrocyte. Transfusion. 1966:6:475–6.

    Article  PubMed  CAS  Google Scholar 

  • Dodge JT, Mitchell C, Hanahan DJ. The preparation and chemical characteristics of hemoglobin-free ghosts. Arch BiochemBiophys. 1963:100:119–29.

    Article  CAS  Google Scholar 

  • Eriksen EF, Danielsen H, Brodersen R. Bilirubin-liposome interaction. Binding of bilirubin dianion, protonization, and aggregation of bilirubin acid. J Biol Chem. 1981;256: 4269–74.

    PubMed  CAS  Google Scholar 

  • Fadok VA, Bratton DL, Frasch SC, Warner ML, Henson PM. The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ. 1998:5:551–62.

    Article  PubMed  CAS  Google Scholar 

  • Fadok VA, de Cathelineau A, Daleke DL, Henson PM, Bratton DL. Loss ofphospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apop-totic cells by macrophages and fibroblasts. J Biol Chem. 2001:276:1071–7.

    Article  PubMed  CAS  Google Scholar 

  • Gascard P, Tran D, Sauvage M, et al. Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane. Biochim Biophys Acta. 1991:1069: 27–36.

    Article  PubMed  CAS  Google Scholar 

  • Glushko V, Thaler M, Ross M. The fluorescence of bilirubin upon interaction with human erythrocyte ghosts. Biochim Biophys Acta. 1982; 719:65–73.

    PubMed  CAS  Google Scholar 

  • Gourley GR. Bilirubin metabolism and kernicterus. Adv Pediatr. 1997:44:173–229.

    PubMed  CAS  Google Scholar 

  • Greenwalt TJ, Dumaswala UJ. Effect of red cell age on vesiculation 171 vitro. Br J Haematol. 1988:68:465–7.

    PubMed  CAS  Google Scholar 

  • Greenwalt TJ, Bryan DJ, Dumaswala UJ. Erythrocyte mem-brane vesiculation and changes in membrane composition during storage in citrate-phosphate-dextrose-adenine-I. Vox Sang. 1984:47:261–70.

    Article  PubMed  CAS  Google Scholar 

  • Hagerstrand H, Holmstrom TH, Bobrowska-Hagerstrand M, Eriksson JE, Isornaa B. Amphiphile-induced phosphatidyl-serine exposure in human erythrocytes. Mol Membr Biol. 1998:15:89–95.

    PubMed  CAS  Google Scholar 

  • Hansen TWR. Acute management of extreme neonatal jaundice-the potential benefits of intensified phototherapy and interruption of enterohepatic bilirubin circulation. Acta Paediatr. 1997:86:843–6.

    Article  PubMed  CAS  Google Scholar 

  • Hansen TWR. Neonatal hyperbilirubinemia and kernicterus. A 1998 update. J Med Liban. 1999:47:22–7.

    PubMed  CAS  Google Scholar 

  • Johnston MV, Hoon AH. Possible mechanisms in infants for selective basal ganglia damage from asphyxia, kernicterus, or mitochondrial encephalopathies. J Child Neurol. 2000; 15:588–91.

    PubMed  CAS  Google Scholar 

  • Kaneko K, Takei Y, Aoki T, Ikeda S, Matsunami H, Lynch S.Bilirubin adsorption therapy and subsequent liver trans-plantation cured severe bilirubin encephalopathy in a long-term survival patient with Crigler-Najjar disease type 1. Intern Med. 2000:39:871–2.

    Google Scholar 

  • Kaul R, Bajpai VK, Shipstone AC, Kaul HK, Krishna Murti CR. Bilirubin-induced erythrocyte membrane cytotoxicity. ExpMolPathol. 1981:34:290–8.

    CAS  Google Scholar 

  • Kirschner-Zilber I, Rabizadeh E, Shaklai N. The interaction of hemin and bilirubin with human red cell membrane. Biochim Biophys Acta. 1982:690:20–30.

    Article  PubMed  CAS  Google Scholar 

  • Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection ofphosphatidylserine expression on B cells under-going apoptosis. Blood. 1994:84:1415–20.

    PubMed  CAS  Google Scholar 

  • Kuypers FA, Lewis RA, Hua M, et al. Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V. Blood. 1996;87:1179–87.

    PubMed  CAS  Google Scholar 

  • Kuypers FA, Yuan J, Lewis RA, et al. Membrane phospholipid asymmetry in human thalassemia. Blood. 1998:91:3044–51.

    PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurements with the Folin phenol reagent. J Biol Chem. 1951:193:265–75.

    PubMed  CAS  Google Scholar 

  • Luly P. Membrane lipid changes in the abnormal red cell. In: Raess BU, Tunnicliff G, eds. The red cell membrane. New Jersey: Clifton; 1989:399–21.

    Google Scholar 

  • Mazurnder S, Sarkar U, Sengupta D. Biochemical profile of erythrocyte membrane of jaundiced neonates. Indian J Exp Biol. 2000:38:91–4.

    Google Scholar 

  • McDonagh AF, Assisi F. The ready isomerization of bilirubin IX-Ot in aqueous solution. Biochem J. 1972:129:797–800.

    PubMed  CAS  Google Scholar 

  • McEvoy L, Williamson P, Schlegel RA. Membrane phospholi-pid asymmetry as a determinant of erythrocyte recognition byrnacrophages. ProeNatlAcad Sci USA. 1986:83:3311–5.

    Article  CAS  Google Scholar 

  • Mireles LC, Lum MA, Dennery PA. Antioxidant and cytotoxic effects of bilirubin on neonatal erythrocytes. Pediatr Res. 1999:45:355–62.

    PubMed  CAS  Google Scholar 

  • Mustafa MG, King TE. Binding of bilirubin with lipid. A possible mechanism of its toxic reactions in mitochondria. J Biol Chem. 1970:245:1084–9.

    PubMed  CAS  Google Scholar 

  • Nagaoka S, Cowger ML. Interaction of bilirubin with lipids studied by fluorescence quenching method. J Biol Chem. 1978:253:2005–11.

    PubMed  CAS  Google Scholar 

  • Odell GB. Neonatal hyperbilirubinemia. New York: Greene & Stratton, Inc; 1980:35–49.

    Google Scholar 

  • Periman JM, Rogers BB, Burns D. Kernicteric findings at autopsy in two sick near term infants. Pediatrics. 1997:99: 612–5.

    Article  Google Scholar 

  • Rashid H, Ali MK, Tayyab S. Differential accessibility of bilirubin to erythrocyte membrane vesicles bearing different structural features. Comp Biochem Physiol Pharmacol Toxicol Endocrinol. 2000:127:345–50.

    Article  CAS  Google Scholar 

  • Rettig MP, Low PS, Gimm A, Mohandas N, Wang J, Christian JA. Evaluation of biochemical changes during in vivo ery-throcyte senescence in the dog. Blood. 1999:93: 376–84.

    PubMed  CAS  Google Scholar 

  • Rimon G, Bazenet CE, Philpott KL, Rubin LL. Increased surface phosphatidylserine is an early marker of neuronal apoptosis. JNeurosciRes. 1997:48:563–70.

    Article  CAS  Google Scholar 

  • Rodrigues CMP, Sola S, Brito MA, Brites D, Moura JJG. Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria. J Hepatol 2002:36:335–41.

    Article  PubMed  CAS  Google Scholar 

  • Rouser G, Fleischer S, Yamamoto A. Two dimensional thin layer chromatographic separation of polar lipids and deter-mination ofphospholipids by phosphorus analysis of spots. Lipids. 1969:5:494–6.

    Google Scholar 

  • Sato H, Aono S, Semba R, Kashiwamata S. Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase-and phospholipase-treated membranes. Biochem J. 1987:248:21–6.

    PubMed  CAS  Google Scholar 

  • Schewe M, Muller P, Korte T, Herrmann A. The role of phospholipid asymmetry in calcium-phosphate-induced fu-sion of human erythrocytes. J Biol Chem. 1992:267:5910–5.

    PubMed  CAS  Google Scholar 

  • Schroit AJ, Madsen JW, Tanaka Y. In vivo recognition and clearance of red blood cells containing phosphatidylserine in their plasma membranes. J Biol Chem. 1985:260:5131–8.

    PubMed  CAS  Google Scholar 

  • Schutte B, Nuydens R, Geerts H, Ramaekers F. Annexin V binding assay as a tool to measure apoptosis in differen-tiated neuronal cells. J Neurosci Methods. 1998:86:63–9.

    Article  PubMed  CAS  Google Scholar 

  • Sharma R, Premachandra BR. Membrane-bound hemoglobin as a marker of oxidative injury in adult and neonatal red blood cells. Biochem Med Metab Biol. 1991:46:33–44.

    Article  PubMed  CAS  Google Scholar 

  • Silva R, Rodrigues CMP, Brites D. Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxy-cholic acid but prevented by ursodeoxycholic acid. J Hepatol. 2001:34:402–8.

    Article  PubMed  CAS  Google Scholar 

  • Tayyab S, Ali MK. Binding of bilirubin to mammalian erythrocytes. Comp Biochem Physiol. 1997;118B:97–103.

    CAS  Google Scholar 

  • Vazquez J, Garcia-Calvo M, Valdivieso F, Mayor F, Mayor F Jr. Interaction of bilirubin with the synaptosomal plasma membrane. JBiol Chem. 1988:263:1255–65.

    CAS  Google Scholar 

  • Verkleij A, Zwaal R, Roelofsen B, Comfurius P, Kastelijn D, van Deenen L. The asymmetric distribution of phospholi-pids in the human red cell membrane: a combined study using phospholipases and freeze-etch electron microscopy. Biochim Biophys Acta. 1973:323:178–93.

    Article  PubMed  CAS  Google Scholar 

  • Vitiello F, Zanetta J-P. Thin-layer chromatography ofphospho-lipids. JChromatogr. 1978:166:637–0.

    Article  CAS  Google Scholar 

  • Zackowski A. Phospholipids in animal eukariotic membranes: transverse asymmetry and movement. Biochem J. 1993:294: 1–14.

    Google Scholar 

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  1. Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, Lisbon, Portugal

    M.A. Brito, R.F.M. Silva & D. Brites

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  1. M.A. Brito
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  2. R.F.M. Silva
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  3. D. Brites
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Brito, M., Silva, R. & Brites, D. Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes. Cell Biol Toxicol 18, 181–192 (2002). https://doi.org/10.1023/A:1015563704551

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