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

ABSTRACT: 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.

SUBMITTER: Melzak KA

PROVIDER: S-EPMC6288636 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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

Melzak Kathryn A KA Uhlig Stefanie S Kirschhöfer Frank F Brenner-Weiss Gerald G Bieback Karen K

Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie 20181030 6

<h4>Background</h4>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.<h4>Methods</h4>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 m ...[more]

PMID: 30574059

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Project description:Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

Dataset Information

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

Publications

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

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