The Blood Bag Plasticizer Di-2-Ethylhexylphthalate Causes Red Blood Cells to Form Stomatocytes, Possibly by Inducing Lipid Flip-Flop

Background: During storage of red blood cell (RBC) concentrates, the plasticizer di-2-ethylhexylphthalate (DEHP) that keeps the blood bags soft leaches out and can be taken up by the RBCs. DEHP is known to be beneficial for the RBC storage quality, but the molecular mechanisms of the action are unknown. Methods: Aqueous suspensions of DEHP were added to RBCs in buffer. The morphological effects were observed on RBCs from 5 donors. Flow cytometry with annexin A5 binding was used to measure the exposed phosphatidylserine. Results: DEHP induced the formation of stomatocytes at concentrations as low as ng/ml, provided that the cell suspension was also sufficiently dilute. Some spherocytes, which were susceptible to lysis, were also formed; after lysis, RBC ghosts were seen to continue the transition to the cup-shaped stomatocyte form. Incubation with DEHP increased the exposed phosphatidylserine, an effect that was also observed in the presence of vanadate, which inhibits the ATP-dependent translocases that maintain the membrane's lipid asymmetry. Conclusions: DEHP can have an active effect on RBC shape, instead of just preventing the storage-related shape changes. The effect appears to be mediated by increased flip-flop of lipids between the leaflets of the RBC membrane.

[1]  Y. Colin,et al.  Spherocytic shift of red blood cells during storage provides a quantitative whole cell–based marker of the storage lesion , 2017, Transfusion.

[2]  D. Devine,et al.  An investigation of red blood cell concentrate quality during storage in paediatric‐sized polyvinylchloride bags plasticized with alternatives to di‐2‐ethylhexyl phthalate (DEHP) , 2016, Vox sanguinis.

[3]  Yanni Xiao,et al.  Eryptosis Indices as a Novel Predictive Parameter for Biocompatibility of Fe3O4 Magnetic Nanoparticles on Erythrocytes , 2015, Scientific Reports.

[4]  D. Devine,et al.  Blood Bag Plasticizers Influence Red Blood Cell Vesiculation Rate without Altering the Lipid Composition of the Vesicles , 2015, Transfusion Medicine and Hemotherapy.

[5]  J. Toca-Herrera,et al.  AFM measurements and lipid rearrangements: evidence from red blood cell shape changes , 2012 .

[6]  R. Nieuwland,et al.  Accumulation of bioactive lipids during storage of blood products is not cell but plasma derived and temperature dependent , 2011, Transfusion.

[7]  C. Lu,et al.  Procoagulant activity of long‐term stored red blood cells due to phosphatidylserine exposure , 2011, Transfusion medicine.

[8]  D. Korte,et al.  DEHP‐plasticised PVC: relevance to blood services * , 2011, Transfusion medicine.

[9]  P. Foster,et al.  Determination of the di-(2-ethylhexyl) phthalate NOAEL for reproductive development in the rat: importance of the retention of extra animals to adulthood. , 2010, Toxicological sciences : an official journal of the Society of Toxicology.

[10]  P. Courtoy,et al.  Three unrelated sphingomyelin analogs spontaneously cluster into plasma membrane micrometric domains. , 2010, Biochimica et biophysica acta.

[11]  Jonathon Howard,et al.  Shapes of Red Blood Cells: Comparison of 3D Confocal Images with the Bilayer-Couple Model , 2008, Cellular and molecular bioengineering.

[12]  M. Kozlov,et al.  How lipid flippases can modulate membrane structure. , 2008, Biochimica et biophysica acta.

[13]  Pakalin Sazan,et al.  European Union Summary Risk Assessment Report - Bis (2-ethylhexyl) Phthalate (DEHP) , 2008 .

[14]  S. Urbaniak,et al.  The application of a new quantitative assay for the monitoring of integrin‐associated protein CD47 on red blood cells during storage and comparison with the expression of CD47 and phosphatidylserine with flow cytometry , 2005, Transfusion.

[15]  Mustafizur Rahman,et al.  The plasticizer market: an assessment of traditional plasticizers and research trends to meet new challenges , 2004 .

[16]  Holger M Koch,et al.  Internal exposure of the general population to DEHP and other phthalates--determination of secondary and primary phthalate monoester metabolites in urine. , 2003, Environmental research.

[17]  Bradley D. Smith,et al.  Facilitated phosphatidylserine (PS) flip-flop and thrombin activation using a synthetic PS scramblase. , 2003, Journal of the American Chemical Society.

[18]  R. Mukhopadhyay,et al.  Stomatocyte–discocyte–echinocyte sequence of the human red blood cell: Evidence for the bilayer– couple hypothesis from membrane mechanics , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[19]  T. Greenwalt,et al.  Biochemical and structural changes in RBCs stored with different plasticizers: the role of hexanol , 2002, Transfusion.

[20]  J. Crowe,et al.  In situ assessment of erythrocyte membrane properties during cold storage , 2002, Molecular membrane biology.

[21]  R. Mukhopadhyay,et al.  Echinocyte shapes: bending, stretching, and shear determine spicule shape and spacing. , 2001, Biophysical journal.

[22]  J. Hess,et al.  The effects of polyvinyl chloride and polyolefin blood bags on red blood cells stored in a new additive solution , 2001, Vox sanguinis.

[23]  J. Boon,et al.  Facilitated phosphatidylcholine flip-flop across erythrocyte membranes using low molecular weight synthetic translocases. , 2001, Journal of the American Chemical Society.

[24]  L. Gray,et al.  The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[25]  S. Bonora,et al.  DSC Study on the Interaction Between Bis-2-(Ethylhexyl) Phthalate and Other o-phthalic Acid Esters and Dipalmitoyl Phosphatidylcholine Liposomes , 2000 .

[26]  C. Högman,et al.  Storage parameters affecting red blood cell survival and function after transfusion. , 1999, Transfusion medicine reviews.

[27]  P. Wong.  A basis of echinocytosis and stomatocytosis in the disc-sphere transformations of the erythrocyte. , 1999, Journal of theoretical biology.

[28]  D. Brooks,et al.  Electrostatic Effects on the Adsorption and Carbodiimide-Mediated Coupling of Ferrichrome A to Amine-Modified Silica , 1995 .

[29]  E. Sackmann,et al.  Budding, fission and domain formation in mixed lipid vesicles induced by lateral phase separation and macromolecular condensation. , 1995, Molecular membrane biology.

[30]  A. Zachowski.  Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. , 1993, The Biochemical journal.

[31]  D. Daleke,et al.  Human erythrocyte shape regulation: interaction of metabolic and redox status. , 1993, Biochimica et biophysica acta.

[32]  M. R. Halie,et al.  Characteristics of red blood cell populations fractionated with a combination of counterflow centrifugation and Percoll separation. , 1992, Blood.

[33]  J. AuBuchon,et al.  The effect of the plasticizer di-2-ethylhexyl phthalate on the survival of stored RBCs. , 1988, Blood.

[34]  W. Reinhart,et al.  Echinocyte‐stomatocyte transformation and shape control of human red blood cells: Morphological aspects , 1987, American journal of hematology.

[35]  R. Labow,et al.  Distribution of di(2-ethylhexyl) phthalate and products in blood and blood components. , 1986, Environmental health perspectives.

[36]  D. Daleke,et al.  Incorporation and translocation of aminophospholipids in human erythrocytes. , 1985, Biochemistry.

[37]  R. Pedersen,et al.  Characterization of erythrocyte quality during the refrigerated storage of whole blood containing di-(2-ethylhexyl) phthalate. , 1984, Blood.

[38]  P. Devaux,et al.  ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J E Ferrell,et al.  Phosphoinositide metabolism and the morphology of human erythrocytes , 1984, The Journal of cell biology.

[40]  A W Jay,et al.  Geometry of the human erythrocyte. I. Effect of albumin on cell geometry. , 1975, Biophysical journal.

[41]  M. Sheetz,et al.  Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[42]  R. Rubin,et al.  Plasticizers from Plastic Devices: Extraction, Metabolism, and Accumulation by Biological Systems , 1970, Science.

[43]  B. Deuticke.  Transformation and restoration of biconcave shape of human erythrocytes induced by amphiphilic agents and changes of ionic environment. , 1968, Biochimica et biophysica acta.

[44]  J. Dodge,et al.  Composition of Phospholipids and of Phospholipid Fatty Acids and Aldehydes in Human Red Cells , 1967 .

[45]  M. Nakao,et al.  Adenosine Triphosphate and Maintenance of Shape of the Human Red Cells , 1960, Nature.