Erythrocyte Shape Changes Research Articles

The effects of non-functionalized polystyrene nanoparticles with different diameters on human erythrocyte membrane and morphology

In this study, the potential toxicity of non-functionalized polystyrene nanoparticles (PS-NPs) in human erythrocytes has been assessed. The effect of PS-NPs with different diameters (∼30 nm, ∼45 nm, ∼70 nm) on fluidity of erythrocytes membrane, red blood cells shape, as well as haemolysis of these cells has been investigated. Erythrocytes were incubated for 24 h with non-functionalized PS-NPs in concentrations ranging from 0.001 to 200 μg/mL in order to study haemolysis and from 0.001 to 10 μg/mL to determine other parameters. Fluidity was estimated by electron paramagnetic resonance (EPR) and the fluorimetric method. It has been shown that PS-NPs induced haemolysis, caused changes in the fluidity of red blood cells membrane, and altered their shape. Non-functionalized PS-NPs increased the membrane stiffness in the hydrophobic region of hydrocarbon chains of fatty acids. The observed changes in haemolysis and morphology were dependent on the size of the nanoparticles. The smallest PS-NPs of ∼30 nm (with the smallest absolute value of the negative zeta potential −29.68 mV) induced the greatest haemolysis, while the largest PS-NPs of ∼70 nm (with the highest absolute value of the negative zeta potential −42.00 mV) caused the greatest changes in erythrocyte shape and stomatocytes formation.

Blood cells and some hematological parameters of red drum (Linnaeus, 1766) in Vietnam.

This research focuses on hematological characteristics, erythrocyte morphology and some biochemical parameters of red drum Sciaenops ocellatus (Perciformes: Sciaenidae), cultured in natural water environment in areas near river mouth (L1), estuaries (L2) in Ha Tinh province and coastal areas (L3) in Nha Trang city, Khanh Hoa province of Vietnam. A total of 18 speciments were examined in research, six in each location. Blood was drawn from the tail vein, using a microscope to research morphology and automated gauges to determine blood biochemical parameters. Analysis of blood samples showed that the rate of red drum's erythrocyte morphology disorders in all three locations was quite high. The two main types of disorders were nuclear deformity and nuclear-matter distribution. Changes in erythrocyte size, shape and nuclear were related to salt concentration at culture locations. Blood hemoglobin content was stable in all three regions. Other hematological parameters such as the number of erythrocytes, blood biochemical parameters (glucose, SGOT, SGPT, urea, creatine, plasma iron, albumin, and protein) have differences among the locations, which showed the different reactions of the same species with different environmental conditions.

Biomarkers for the central nervous system complications of sickle cell disease: are we there yet?

Sickle cell disease (SCD, OMIM #603903), an autosomal recessively inherited β-hemoglobinopathy, was the first human disorder delineated at a molecular level. The putative single nucleotide mutation in the HBB gene generates an abnormal hemoglobin species, which polymerizes in deoxygenated conditions causing irreversible changes in erythrocyte shape and function. Sickling erythrocytes are in turn responsible for microvascular vaso-occlusion, hemolysis and a systemic vasculopathy in patients. SCD has represented an attractive field for proteomic investigation since its methodological infancy. Clinically actionable biomarkers, especially for the prevention of cerebrovascular complications in children with the condition, are urgently needed and their discovery remains a major challenge. In this issue, Lance and colleagues report of their unbiased proteomic studies on samples from the participants of the landmark prospective, randomized, single-blind SIT trial (NEJM 2014). Their results reveal numerous brain-enriched plasma proteins specific for SCD, and for silent cerebral infarcts in this disorder, and further analyses highlight novel cellular mechanisms behind the brain damage in SCD. Although the goal of identifying reliable biomarker candidates for cerebrovascular complications could not be met, the dataset produced by the authors constitutes a significant contribution to the field and opens new horizons for further clinical and laboratory investigation.

RETRACTED: Toxicity of polystyrene nanoplastics in Ctenopharyngodon idella juveniles: A genotoxic, mutagenic and cytotoxic perspective

The increased contamination of surface water with plastic waste is proportional to the increased consumption of products that use them as raw material. However, the impact of these residues on aquatic biota remains limited, mainly when it comes to nanoplastics (NPs). Thus, the aim of the current study is to test the hypothesis that the exposure of Ctenopharyngodon idella juveniles to polystyrene nanoplastics (PS NPs) at low concentrations (0.04 ng/L, 34 ng/L and 34 μg/L), for 20 days, leads to DNA damage and has mutagenic and cytotoxic effects on their erythrocytes. Comet assay enabled observing that DNA damage (inferred from the greater tail length, DNA percentage in the tail and Olive tail moment) induced by PS NPs has increased as the pollutant concentrations have increased, as well as that the formation of micronuclei and other nuclear abnormalities was equitable in animals exposed to this pollutant. On the other hand, there were significant changes in erythrocyte shape and size, oxidative stress generation (NO levels, lipid peroxidation, hydrogen peroxide), antioxidant system inhibition (mediated by total hepatic glutathione) and PS NPs accumulation in the liver and brain of animals exposed to higher concentrations of it. Therefore, the current study has confirmed the initial hypothesis and enhanced the knowledge about the genotoxic, mutagenic and cytotoxic potential of PS NPs in freshwater fish at early developmental stage, relating these effects to biochemical changes and significant accumulation of these nanomaterials. Besides, it is a warning about the (eco) toxicological risk represented by these nanopollutants in aquatic environments. CapsulePolystyrene nanoplastics are capable of inducing DNA damage, mutagenic and cytotoxicity changes in fish.

Shape changes of erythrocytes during blood clot contraction and the structure of polyhedrocytes

Polyhedral erythrocytes, named polyhedrocytes, are formed in contracted blood clots and thrombi, as a result of compression by activated contractile platelets pulling on fibrin. This deformation was shown to be mechanical in nature and polyhedrocytes were characterized using light and electron microscopy. Through three-dimensional reconstruction, we quantified the geometry of biconcave, intermediate, and polyhedral erythrocytes within contracting blood clots. During compression, erythrocytes became less oblate and more prolate than the biconcave cells and largely corresponded to convex, irregular polyhedra with a total number of faces ranging from 10 to 16. Faces were polygons with 3 to 6 sides. The majority of the faces were quadrilaterals, though not all sides were straight and not all faces were flat. There were no changes in the surface area or volume. These results describe the gradual natural deformation of erythrocytes as a part of compaction into a tightly packed array that is an important but understudied component of mature blood clots and thrombi.

Ischemic Pain Testing Demonstrates Hypoxia Dependent Early Pain and Hypoxia Independent Persistent Pain Responses in Adults with Sickle Cell Anemia

The hallmark manifestation of homozygous sickle cell disease (SCD) is acute pain. The underlying etiology is unknown. It is believed that hypoxia induces changes in erythrocyte shape and produces microvasculature occlusion by sickled red cells. Vascular occlusion is believed to represent the event inducing tissue hypoxia, reperfusion injury and pain.Studies have only recently characterized pain responses to experimental stimuli in rodents and humans with SCD using quantitative sensory testing (QST). SCD is characterized by thermal and mechanical sensitivity, and there is evidence of central sensitization (heightened sensitivity to pain) from QST, temporal summation and functional MRI studies. However, the effect of hypoxia upon nociception has not been studied despite the need for hypoxia to induce sickle hemoglobin polymerization. The ischemic tourniquet pain test is a QST used to investigate nociception. It was the first QST to demonstrate opioid analgesia. While it is assumed that the resulting pain is due to hypoxia, no studies have demonstrated the degree of hypoxia needed to produce pain.Methods: Thirty adults with SCD and 30 age and sex matched 30 controls underwent the ischemic pain test. A pneumatic cuff was inflated around the upper arm to induce forearm hypoxia, and the time from cuff inflation to first reported pain (pain threshold) and until pain became intolerable (pain tolerance) was measured. Pain was quantified on a 20 point scale. Time to pain threshold and tolerance were the primary end points. Testing was repeated after a washout period in a subset of the initial subjects: 18 SCD patients and 22 controls. Repeat testing proceeded with a 15 minute observation period followed by the ischemic pain test with the time and pain score recorded at threshold and tolerance. Subjects were monitored after cuff deflation during recovery with pain scores obtained at 30 seconds, then every minute for 5 minutes and finally every 3 minutes over a total recovery period of 20 minutes. Skeletal muscle tissue oxygenation at the thenar eminence was measured continuously with a near infrared spectroscopy (NIRS) monitor (OxiplexTS; ISS, Champaign, IL).Results: SCD subjects reached the pain threshold at a mean of 6.44 versus 11.57 minutes in controls (p=0.07). Time to pain tolerance was significantly different (SS 11.29 versus controls 19.59 minutes; p=0.004). Both groups reported identical pain scores. By stepwise linear regression, SCD (p=0.002) and gender (p=0.0008) were associated with time to pain tolerance (r2=0.29), while recent opioid use and pain crisis frequency were not. Because these results demonstrated that SCD adults reach tolerance more rapidly than controls, we sought to determine if this difference was due to tissue hypoxia. Pain scores with repeat testing were not significantly different at threshold or tolerance. However, pain score curves over testing and recovery were significantly different (p=0.003, Fig 1A). SCA had pain above baseline after 11 minutes of recovery (p=0.02) suggesting a persistent pain response compared to controls. Oxyhemoglobin and deoxyhemoglobin from NIRS were not different between groups at the primary endpoints (Fig 1B and 1C). StO2 (% tissue oxygenation, Fig 1D) at both threshold and tolerance was not different between SCD and controls, suggesting that the onset and maximal level of pain during this QST is defined an StO2 thresholds 40% common to sickle cell patients and controls. However, oxyhemoglobin, deoxyhemoglobin and StO2 had already recovered to baseline by the time the pain was evident (Fig 1B, 1C and 1D).Conclusions: Adults with SCD reach hypoxia induced, experimental pain earlier than NVs. Time to maximal pain is associated with SCD and sex. NIRS showed that pain tolerance occurred at a uniform tissue oxygenation threshold indicating hypoxia is the primary determinant for sensing acute pain. However, persistent SCD pain was independent of tissue oxygenation, and could be explained by the presence of peripheral or central sensitization. These data suggest that SCD pain treatment may require improved oxygen delivery to affected tissues and analgesics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa).

Endemic Sicilian pond turtles Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink were examined for the presence of haemogregarine parasites. The presence of haemogregarines, occurring mainly in the microgametocyte stage (13.2 ± 0.12 μm in length and 6.4 ± 0.52 μm in width), was observed in approximately 9% of the sampled E. trinacris. Based on the observed morphology and on the sequencing of nuclear 18S rDNA, we identified the parasite as Haemogregarina stepanowi Danilewsky, 1885. Morphometric study of uninfected and infected red blood cells has shown that H. stepanowi induces different changes in erythrocyte shape depending on the infective stage. The differential count of leukocytes in specimens infected with H. stepanowi showed no significant difference compared with healthy specimens. However, considering the health problems which might be induced by H. stepanowi in the closely related European pond turtle Emys orbicularis (Linneaus), monitoring of the health status of the infected Sicilian populations of E. trinacris is desirable. The restricted distribution of populations of Emys infected with haemogregarines in Sicily is quite puzzling and the possible human-mediated introduction of the parasite in Sicily is briefly discussed.

Imaging flow cytometry for automated detection of hypoxia‐induced erythrocyte shape change in sickle cell disease

In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = -0.558, P = 0.027), negatively with pH (R = -0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = -0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.

Radiographic contrast media alterate the localization of actin/band4.9 in the membrane cytoskeleton of human erythrocytes.

Different radiographic contrast media (RCM) were shown to induce morphological changes of blood cells (e.g. erythrocytes or thrombocytes) and endothelial cells. The echinocytic shape change of erythrocytes, particularly, affords alterations of the membrane cytoskeleton. The cytoskeleton plays a crucial role for the shape and deformability of the red blood cell. Disruption of the interaction between components of the red blood cell membrane cytoskeleton may cause a loss of structural and functional integrity of the membrane. In this study band4.9 and actin as components of the cytoskeletal junctional complex were examined in human erythrocytes after suspension in autologous plasma or in plasma RCM mixtures (30% v/v Iodixanol-320 or Iopromide-370) followed by a successive double staining with TRITC-/FITC-coupled monoclonal antibodies. After adding Iopromide-370 to the plasma in practically none of the cells the rounded conformation of the membrane cytoskeleton - as it appeared in cells suspended in autologous plasma - was found. In addition, Iopromide-370 induced thin lines and coarse knob-like structures of band4.9 at the cell periphery while most cell centers were devoid of band4.9, and a box-like arrangement of bands of band4.9. A dissociation between colours red (actin) and green (band4.9) occurred as well. In contrast, erythrocytes suspended in a plasma/Iodixanol-320 mixture showed a membrane cytoskeleton comparable to cells suspended in autologous plasma, Similar results were found with respect to the distribution of actin. This study revealed for the first time RCM-dependent differences in band4.9 activities as possible pathophysiological mechanism for the chemotoxicity of radiographic contrast media.

Polyhedrocytes: Compressed Polyhedral Erythrocytes In Contracted Blood Clots and Thrombi

Background Contraction of blood clots is necessary for hemostasis, wound healing and to restore flow past obstructive thrombi. However, little has been known about the structure of contracted clots and mechanisms of contraction. Erythrocytes, biconcave cells that are highly deformable to allow their passage through the microvasculature, are abundant in venous thrombi, and to a lesser extent in arterial thrombi. Erythrocytes promote hemostasis, but their participation in clot contraction has not been reported. Here we study the mechanisms of clot contraction and the roles of erythrocytes, platelets and fibrin, and show that erythrocyte shape change into compressed polyhedrocytes allows tight packing consistent with the major function of clots to stem bleeding. Methods Whole blood was clotted by recalcification and addition of thrombin or kaolin, while following the process of clotting, including contraction, with a new technique using T2 magnetic resonance. We examined the structure and composition of contracted whole blood clots by scanning electron microscopy and confocal light microscopy. Results Contracted clots display a remarkable structure, with a close-packed, tessellated array (or mosaic tiling of space) of compressed polyhedral erythrocytes (called polyhedrocytes) on the interior and a meshwork of fibrin and platelet aggregates on the exterior. Little fibin and few platelets were found on the interior of the contracted clots. The same results were obtained with both thrombin and kaolin as activators of clotting and also with reconstituted human blood and clots prepared from mouse blood. Confocal microscopy of hydrated clots confirms the results of scanning electron microscopy. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a closely packed polyhedral array. To assess the density of packing of the polyhedral erythrocytes, we replaced the water surrounding the clots with D2O and observed by T2 magnetic resonance that hydrogen/deuterium exchange for the contracted clots was very slow, consistent with their very tightly packed, almost impermeable structure. The same polyhedrocyte structures were observed from in vivo thrombi aspirated by cardiologists from the coronary arteries of ST-elevation myocardial infarction patients. Summary/Conclusions We have observed a previously undiscovered, naturally occurring erythrocyte function and morphology, closely packed polyhedra, in contracted clots and thrombi, and an unexpected spatial redistribution of platelets and fibrin that occurs during contraction. Clot contraction is an essential part of hemostasis, since both human genetic disorders of platelet myosin IIA and megakaryocyte myosin IIA-knock out mice show a bleeding phenotype. These observations on contracted clots imply that they are stiff, rigid structures that can form an impermeable, watertight seal. On the one hand, contraction of clots within the vasculature may relieve obstruction of blood vessels and allow recanalization, especially in the venous system. On the other hand, these results account for long-standing clinical observations that fibrinolysis is greatly prolonged following clot contraction, since perfusion or diffusion of lytic enzymes into these tightly packed polyhedral erythrocytes would be nearly impossible. These results suggest a vital role for erythrocytes and clot contraction in hemostasis and wound healing. Disclosures: No relevant conflicts of interest to declare.

Distribution of actin of the human erythrocyte membrane cytoskeleton after interaction with radiographic contrast media

A type-dependent chemotoxic effect of radiographic contrast media on erythrocytes and endothelial cells was reported several times. While mechanisms of toxicity are still unclear the cellular reactions e.g. echinocyte formation in erythrocytes and the buckling of endothelial cells coincided with deterioration of capillary perfusion (in patients with coronary artery disease) and tissue oxygen tension (in the myocardium of pigs). Whether the shape changes in erythrocytes coincide with changes in the arrangement of actin, the core of the actin-spectrin cytoskeletal network and possible actor in membrane stresses and deformation is not known until now. To get specific informations actin was stained using two different staining methods (antibodies to β-actin staining oligomeric G-actin and polymeric F-actin and Phalloidin-Rhodamin staining polymeric F-actin only). In addition, an advanced version of confocal laser scanning microscopes was used enabling the display of the actin arrangement near substrate surfaces. Blood smears were produced after erythrocyte suspension in autologous plasma or in two different plasma/RCM mixtures. In this study an even homogenous distribution of fine grained globular actin in the normal human erythrocyte could be demonstrated. After suspension of erythrocytes in a plasma/Iodixanol mixture an increased number of membrane protrusions appeared densely filled with intensely stained actin similar to cells suspended in autologous plasma, however, there in less numbers. Suspension in Iopromide, in contrast, induced a complete reorganization of the cytoskeletal actin: the fine grained globular actin distribution disappeared and only few, long and thick actin filaments bundled and possibly polymerized appeared, instead, shown here for the first time.

Effect of Laser Irradiation of Donor Blood on Erythrocyte Shape

Changes in erythrocyte shape in donor blood during storage and after irradiation with He-Ne laser and infrared laser were studied by scanning electron microscopy, thick drop express-method, and morphometry. It was found that laser irradiation delayed the appearance of erythrocytes of pathological shapes (echinocytes, stomatocytes, etc.) in the blood; He-Ne laser produced a more pronounced effect.

The location of organotins within the erythrocyte membrane in relation to their toxicity

The aim of the present study on organotin compounds, which are toxic to biological systems, was to determine the relationship between the compounds’ toxicity and their location in the lipid bilayer of the biological membrane. It was assumed that the degree of disturbance caused within the lipid bilayer of the membrane, which in turn depends on the depth of incorporation, was an appropriate measure of toxicity.Previous results from our studies on the effect of organotin chlorides on membranes, made by using infrared radiation and hemolysis of erythrocytes, indicated that tributyltin (TBT) is the most active in terms of its interaction with the erythrocyte membrane. This compound causes the most severe hemolysis of erythrocytes and dehydration of membrane constituents. In order to connect the changes induced within the membrane structure with the compounds’ location, we have investigated erythrocyte shape changes using both microscopic and fluorimetric methods. The microscopic results show that organotin compounds accumulate in the outer monolayer of the membrane. The fluorimetric studies indicate that all the compounds are present in the hydrophilic part of the outer lipid monolayer, and change the order parameter of the layer. However, only tributyltin, by being incorporated into the hydrophobic region of the monolayer, changes the fluidity in the alkyl chain region of the erythrocyte membrane.Furthermore, only TBT is present in both the hydrophilic and hydrophobic regions, as evidenced by the changed order parameter of the polar groups and fluorescence anisotropy of DPH probe in the hydrophobic region, these being connected with its high toxicity.

A study of the properties of erythrocytes with polarized light under hypotonic conditions

We consider the application of polarized radiation to the study of the properties of blood and erythrocyte suspensions, including the case of disturbance of the osmotic balance in the erythrocyte-surrounding-medium system. We study experimentally erythrocyte suspensions prepared by diluting whole donor blood with autoplasma, serum, and NaCl aqueous solutions of different concentrations. The Stokes parameters of scattered radiation have been measured with a Stokes polarimeter using a He-Ne laser as a source of linearly or elliptically polarized radiation (λ = 0.63 μm). We show that, upon illumination of erythrocyte suspensions with linearly or circularly polarized radiation, the Stokes parameters of the scattered radiation are highly sensitive to changes in the shape and size of erythrocytes upon hypotonic swelling.

Evaluation of the effect of Uncaria tomentosa extracts on the size and shape of human erythrocytes (in vitro)

In this study, we continued our investigations concerning the interaction of Uncaria tomentosa extracts with the human erythrocytes. The analysis of the size and shape of the erythrocytes by means of flow cytometry and phase contrast microscopy was performed. We executed our experiments using ethanolic and aqueous extracts from the leaves and bark of U. tomentosa. Disturbances were observed in the size and shape of the erythrocytes incubated with ethanolic and aqueous extracts at the concentrations of 100μg/mL and 250μg/mL, respectively. The observed changes were probably related to the entry of polyphenolic compounds contained in U. tomentosa extracts into erythrocyte membrane. Externalization of phosphatidylserine on the erythrocytic surfaces was also noticed during incubation with extracts at concentration of 250μg/mL. We concluded that all of the extracts examined induced changes in the erythrocyte membrane properties, whereas ethanolic extracts from bark induced the most significant changes. The possible binding of polyphenols to the erythrocyte surface may have accounted for the protective properties of extracts against haemolysis of RBCs, which was observed in our previous study (Bors et al., 2011), but considerable incorporation of polyphenols into cell membranes can result in disturbance of phosphatidylserine transport and changes in erythrocyte shape. Nevertheless the results of the investigations showed that considerable morphological changes appear only as a result of erythrocyte exposure to high concentrations (50ppm and 100ppm) of the extracts studied, thus they should not lead to clinical erythrocytic damage if recommended doses of U. tomentosa preparations are administrated.

Low Level of Lead Can Induce Phosphatidylserine Exposure and Erythrophagocytosis: A New Mechanism Underlying Lead-Associated Anemia

Anemia is probably one of the most well-known toxic effects of lead. Previously, lead-induced anemia was considered to be from the inhibition of δ-aminolevulinic acid dehydratase participating in the heme biosynthesis. However, little is known whether lead could affect the destruction of erythrocyte, another important factor for anemia. In the present study, we demonstrated that lead could accelerate the splenic sequestration of erythrocytes through phosphatidylserine (PS) exposure and subsequently increased erythrophagocytosis. In freshly isolated human erythrocytes, Pb(2+)- induced PS exposure at relatively low concentrations (∼0.1 μM) by inhibiting flippase, a key aminophospholipid translocase for the maintenance of PS asymmetry and adenosine triphosphate depletion appeared to underlie this phenomenon. Abnormal shape changes of erythrocytes and microvesicle generation and other triggers for the erythrophagocytosis were also observed in the Pb(2+)-exposed erythrocytes. In vitro data showed that human macrophage indeed recognized and phagocytosis PS-exposed erythrocytes. In good accordance with these in vitro results, the oral administration of Pb(2+) increased PS exposure on erythrocytes in rat in vivo. In addition, reduction of hematocrit and hemoglobin and increased spleen weight were observed along with enhanced splenic sequestration of erythrocytes in the rats exposed to Pb(2+) subchronically for 4 weeks through drinking water. In conclusion, these results suggest that Pb(2+)-induced anemia may be explained at least in part by increased PS exposure on erythrocytes, erythrophagocytosis, and splenic sequestration.

Reconstruction of erythrocyte shape during modified morphological response

Changes in erythrocyte shape during morphological response modified by benzalkonium chloride (BzA) were studied in sucrose solutions. Fixation of the cells with glutaraldehyde- and formaldehyde-containing fixatives at some time points is usually inadequate to maintain the current cell shape. Considering the reconstruction of erythrocyte shape, which takes into account the mode of fixative action, we showed that the echinocyte-forming activity of BzA depends on the concentration of this surfactant. It can induce a direct spherostomatocyte-spheroechinocyte transition without altering the near-spherical shape of the cells. On the other hand, the reverse spheroechinocyte-spherostomatocyte transition was always accompanied by some flattening of the cells, although in some instances discoidal shape was not achieved. The data point to asymmetric shape transitions of erythrocytes in sucrose solution, which contradicts the continuum and bilayer-couple models of shape regulation. It seems that the nonuniform structure of native erythrocyte membrane plays a more important role in morphological transitions of these cells than suggested earlier.

Effect of phosphatidylcholine chlorohydrins on human erythrocytes

Hypochlorite generated in vivo under pathological conditions is a known oxidant and chlorinating agent, able to react with proteins and lipids, which affects the stability of biological membranes. Reaction with unsaturated fatty acyl chains in glycerophospholipids such as phosphatidylcholine results in the formation of chlorohydrins. The aim of this study was to determine the effects of chlorohydrins formed by the reaction of hypochlorite with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonylphosphatidylcholine on biophysical properties of bilayers and their effects on human erythrocytes. Using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in a decrease in the rotational correlation time and an increase in the order parameter of liposomes. Unilamellar chlorohydrin liposomes had a lower permeation coefficient for calcein than liposomes made of parent lipids. Flow cytometry demonstrated fast incorporation of uni and multilamellar chlorohydrin liposomes labeled with NBD-phosphatidylethanolamine into erythrocytes. This effect was accompanied by changes in erythrocyte shape (echinocyte formation) and aggregation. Similar but less pronounced effects were noticed for parent lipids only after longer incubation. Chlorohydrins showed also a stronger hemolytic action, proportional to the lipid:erythrocyte ratio. These results are important for understanding the effects of HOCl on mammalian cells, such as might occur in inflammatory pathology.

The effect of membrane diffusion potential change on anionic drugs indomethacin and barbitone induced human red blood cell shape change and on cellular uptake of drugs

The effect of membrane potential change on anionic drugs Indomethacin and barbitone induced human erythrocyte shape change and red cell uptake of drug has been studied using microscopy and spectrophotometry techniques respectively. The membrane potential was changed by reducing the extracellular chloride concentration while maintaining the ionic strength and the osmolarity of the cell suspending solutions constant. At indomethacin and barbitone concentractions necessary to cause marked cell crenation membrane diffusionpotential change from - 7.1 mV to 16 . 4niV, led to the reversal of the shape changing properly of the drugs to cup formers. The cellular uptake of drug increased with increasing membrane potential. The membrane potential dependent shape change was also reversible on revering the membrane potential. The results suggest that cellular uptake of drug and drug induced cell shape change wasstrongly dependent on changes in the extracellular chloride concentration which alter the potential across the erythrocyte membrane. These findings may be of medical significance such as in the design of drugs, for example, for sickle cell diseases, malarial diseases, since knowledge of the concentration and location of the molecules with respect to the membrane surface is required.

Membrane potential change effects on cationic and neutral drug - induced erythrocyte shape change and cellular uptake of drugs

The effect of membrane potential change of the human erythrocytes on cationic drugs tetracaine and chlorpromazine and neutral drug benzyl alcohol induced cell shape change and red cell uptake of drug has been quantitated using light microscopy and spectrophotometry respectively.At the drug concentration necessary to cause cell membrane cell shape change membrane potential change from -7.1 mV to 1 6.4mV let to the reversal of the cup-forming property of chlorpromazine and tetracaine to that of a crenetor at both 20°C and 37°C. The effect of alteringthe membrane potential from -7. lmV to 16.4mV also led to the decrease of cellular uptake of drug with increasing membrane diffusion potential. The membrane potential dependent drug induced cell shape change with also reversible on reversing the membrane potential. The results therefore suggest that the cellular uptake of drug and drug induced cell shape change in human erythrocytes was dependent on change in extracellular chloride concentration