Hypotonic Hemolysis Research Articles

Evaluation of Dose-dependent Interactions of Antibiotics using RBC Membrane Model Systems

Background: The present study aims to understand the membrane interactions of three different antibiotics widely used for non-specific viral diseases. Methods: The dose-dependent membrane interactions of the selected antibiotics were elucidated using octanol: water partition coefficient and hemolysis assay. Results: Our octanol: water partition coefficient experiments indicated that among the three antibiotics (Azithromycin, Erythromycin, and Amoxicillin), azithromycin and erythromycin were more hydrophobic compared to amoxicillin. This finding also correlated with hypotonic hemolysis assay experiments where the membrane stabilization effects of both azithromycin and erythromycin were reduced in a dose-dependent manner compared to amoxicillin. Conclusion: This may be attributed to the hydrophobic nature of azithromycin, and erythromycin penetrates deeper into the RBC membrane to the extent of the hydrophobic core compared to amoxicillin. The result is also concurrent with the observations from the hemolysis assay in the isotonic conditions. To conclude, among the three antibiotics, azithromycin, erythromycin localized on the hydrophobic core of the lipid membrane, and amoxicillin localized on the outer surface and slight penetration to the outer leaflet of the membrane.

Evaluation of the Anti-inflammatory and Anti-hemolytic Potential of Polyphenolic Components of Common Mallow (Malva sylvestris)

Background: This work aims at the assessement of anti-inflammatory and anti-hemolytic effect of Malva sylvestris. Methods: The anti-inflammatory potential was evaluated by the inhibition of protein denaturation method. It was followed by the study of anti-hemolytic potential, based on two methods (haemolysis by hydrogen peroxide (H2O2) and by hypotonic haemolysis). Result: The macerated aqueous extract of M. sylvestris (250 µg/ml) exhibited the highest inhibition percentage of BSA denaturation compared to other extracts but it appeared to be slightly lower than the drug diclofenac sodium (80.97±1.23%). On the other hand, the macerated aqueous extract showed more protective power against haemolysis (93.42±3.45%). While it was almost similar to the percentage recorded for ascorbic acid (93.68±3.21%). For the second method, it was observed that the decocted acetone extract of M. sylvestris showed a rate of haemolysis inhibition which was the highest (98.09±1.26%) but that it remained slightly lower than aspirin (98.77±0.44%). All of these results showed that M. sylvestris extracts have interesting anti-inflammatory and anti-haemolytic potential and therefore have considerable interest as an alternative treatment against inflammatory mechanisms.

Correlation between sphingomyelin and the membrane stability of mammalian erythrocytes.

Lipid compositions of mammalian erythrocyte membranes are different among species. Therefore, the information on hemolysis from mammalian erythrocytes is useful to understand membrane properties of human erythrocytes. In this work, pressure-induced hemolysis and hypotonic one were examined using erythrocytes of human, sheep, cow, cat, dog, pig, horse, rat, and mouse. Pressure-induced hemolysis was suppressed by membrane sphingomyelin, whereas hypotonic hemolysis decreased upon increment of cell diameter. Mass spectra of erythrocyte membrane lipids demonstrated that sphingomyelin with an acyl chain 24:1 was associated with the suppression of pressure-induced hemolysis. In cow erythrocytes, pressure-induced hemolysis was greatly suppressed and the detachment of cytoskeletal proteins from the membrane under hypotonic conditions was also inhibited. Taken together, these results suggest that sphingomyelin with 24:1 fatty acid plays an important role in the stability of the erythrocyte membrane, perhaps via cholesterol.

Assessment of membrane stability, central nervous system depressant, and gut motility effects of Lablab purpureus seeds

Introduction: Lablab purpureus, under the family of Fabaceae, is a plant with various pharmacological activities. The present study was aimed to investigate the phytoconstituents, membrane stabilizing activity, central nervous system (CNS) depressant potential, and gastrointestinal (GI) motility of the methanol extract of L. purpureus seeds (MELPS).Methods: The methanol plant extract was screened for different phytochemical groups. Mice were classified into four groups for in vivo activities. Group-I was designated as negative control and received distilled water (10 mL/kg body weight); group-II served as positive control and received diazepam (1 mg/kg body weight). Group-III and group-IV both were experimental groups and received plant extract at 200 and 400 mg/kg body weight, respectively.Results: Alkaloids, carbohydrates, saponins, glycosides, tannins, phenols, flavonoids, and proteins were found after phytochemical analysis. On hypotonic solution-induced hemolysis of erythrocyte membrane, MELPS9 (9 mg/mL) resulted in the highest percentage of inhibition (60.51 ± 0.889), and on heat-induced hemolysis, MELPS9 (9 mg/mL) resulted in the highest percentage of inhibition (33.97 ± 0.21). In the case of the CNS depressant potential experiment, mice that received a sample at a dose of 400 mg/kg body weight showed the highest result (54.40 ± 4.51) compared with the positive control (14.2 ± 3.70) (P < 0.001). Similarly, 400 mg/kg dose sample exhibited the highest percentage of inhibition (60.51 ± 0.889) of hemolysis and GI motility (22.26%).Conclusion: It can be concluded that the MELPS has potential membrane stability, CNS depressant, and antimotility effects.

State of antioxidant system in urogenital trichomoniasis and membranotropic effect of metronidazole

The objective of this work was to study the activity of glutathione peroxidase, glutathione reductase and the level of sulfhydryl groups in erythrocytes of patients with urogenital trichomoniasis and the effect of metronidazole on the degree of osmotic and peroxide resistance of erythrocytes from healthy donors. We examined 15 patients with urogenital trichomoniasis and 20 healthy volunteers. We studied native preparations, and also carried out a culture method using the Johnson-Trussel nutrient medium (CPLM) to identify Trichomona vaginalis. The activity of glutathione reductase, glutathione peroxidase and the level of total sulfhydryl groups were determined in erythrocytes of peripheral blood. The membrane effect of metronidazole was evaluated in in vitro experiment by the degree of osmotic and peroxide resistance of erythrocytes from healthy people. It has been established that a significant decrease in glutathione reductase and glutathione peroxidase activities in erythrocytes is observed, which indicates a violation of the antioxidant system in this pathology. It was shown in vitro experiment, that metronidazole in low concentration (80 μmol /l) has the ability to inhibit erythrocyte hypotonic hemolysis, and high concentration (250 μmol/l) leads to a decrease in osmotic and peroxide resistance of erythrocytes. Thus, inhibition of the activity of the enzymatic link of the antioxidant defense is observed in urogenital trichomoniasis, which is one of the mechanisms for the development of pathology at the cellular level in this disease. It has been shown that the isolated membranotropic action of metronidazole depends on its concentration – the drug at low concentration is able to inhibit hypotonic hemolysis of erythrocytes, and high concentration makes them more sensitive to the osmotic and peroxide hemolysis. The obtained results should be taken into account in the development of complex methods of therapy for urogenital trichomoniasis.

Extraction optimization of bioactive compounds from ora-pro-nobis (Pereskia aculeata Miller) leaves and their in vitro antioxidant and antihemolytic activities

Ora-pro-nobis (Pereskia aculeata Miller) is a non-conventional food plant common in Brazil. The objective of this study was to optimize the extraction of bioactive phenolic compounds from ora-pro-nobis leaves by employing solvent mixtures. Ten extracts were obtained with water, ethanol, acetone, and their binary and ternary mixtures, evaluating the chemical composition, antioxidant activity and bioactivities in vitro. The response surface methodology was applied to model the results and calculate the optimal solvent composition, which is 60% water, 40% ethanol and 0% acetone. The optimized extract is rich in phenolic compounds (64 mg GAE/g) and proteins (823 mg/g) and presents antioxidant activity (in intracellular media as well) and inhibits lipid peroxidation (32%) along with hypotonic hemolysis (H50 = 0.339%), it does not present toxicity in vitro against cancer and normal cells. This is the first report of chicoric, caffeoyl-hexaric and coumaroyl-hexaric acids and some glycosylate derivatives of flavonols in ora-pro-nobis leaves.

Antioxidant, antinociceptive, anti-inflammatory, and hepatoprotective activities of pentacyclic triterpenes isolated from Ziziphus oxyphylla Edgew

Ziziphus oxyphylla Edgew is in folk use in Pakistan as an analgesic, anti-inflammatory, and liver ailments. Therefore, we have investigated antioxidant, antinociceptive, anti-inflammatory, and hepatoprotective activities of the isolated compounds (ceanothic acid and zizybrenalic acid) from the chloroform fraction of Z. oxyphylla. Ceanothic acid and zizybrenalic acid showed significant DPPH and H2O2 scavenging activity as compared to control. In the acute toxicity study, ceanothic acid and zizybrenalic acid showed no toxic effects upto 200 mg/kg. The antinociceptive activity shown by ceanothic acid and zizybrenalic acid at 50 mg/kg was 64.28% and 65.35% compared to diclofenac sodium (72.3%) at 50 mg/kg. The percent inhibition of xylene-induced ear edema exhibited by ceanothic acid and zizybrenalic acid at 50 mg/kg was 51.33% and 58.66%, respectively, as compared to diclofenac sodium (72.66%). Both the isolated compounds exhibited inhibition of carrageenan-induced paw edema as compared to control. Hepatoprotection exhibited by zizybrenalic acid was more pronounced than ceanothic acid as observed from the decrease in carbon tetrachloride (CCl4)-induced elevation of serum biomarkers, antioxidant enzymes and lipid peroxidation. Furthermore, zizybrenalic acid produced a marked decline in CCl4-induced prolongation of phenobarbital-induced sleeping duration. Zizybrenalic acid exhibited 55.4 ± 1.37% inhibition of hypotonic solution-induced hemolysis compared to sodium salicylate (75.6 ± 2.15%). The histopathological damage caused by CCl4 was also countered by the administration of ceanothic acid and zizybrenalic acid. Ceanothic acid and zizybrenalic acid exhibited antioxidant, antinociceptive, anti-inflammatory, and hepatoprotective activities. Zizybrenalic acid exhibited better antioxidant, antinociceptive, anti-inflammatory, and hepatoprotective activity than ceanothic acid.

Effects of Cholesterol on Membrane Stability of Human Erythrocytes.

Human erythrocytes contain abundant cholesterol as membrane lipids. Cholesterol contributes to the stability and function of the membrane. Membrane stability of the erythrocyte has been mainly examined under hypotonic conditions, but not under high hydrostatic pressure. So, the effect of cholesterol on the membrane stability of human erythrocyte was examined under a pressure of 200 MPa. As with hypotonic hemolysis, the pressure-induced hemolysis was enhanced by depletion of cholesterol from the intact erythrocyte membrane, whereas suppressed by cholesterol loading to the intact one. Enhancement of such hemolysis was associated with the suppression of fragmentation, whereas the hemolysis was suppressed by the facilitation of vesiculation. Cholesterol induced the tight linkage of the lipid bilayer with cytoskeleton. Taken together, these results suggest that the erythrocyte membrane stability is affected by such tight linkage by cholesterol.

In vitro and in vivo evaluation of pharmacological potentials of Campsis radicans L

BackgroundCampsis radicans L. is a flowering plant in Bangladesh, traditionally used for the treatment of several human diseases. In this study, in vitro antioxidant, thrombolytic, membrane stabilizing and in vivo analgesic, hypoglycemic, anti-diarrheal and CNS antidepressant activities of organic soluble fractions of crude methanol extract of C. radicans leaf were investigated using appropriate experimental models.MethodsThe leaves of C. radicans were collected, authenticated, dried and extracted with methanol at room temperature for 30 days. The concentrated methanol extract was partitioned to petroleum-ether (PESF), dichloromethane (DMSF) and ethyl acetate (EASF) soluble fractions. The antioxidant activity of these fractions was determined by DPPH free radical scavenging method. Total phenolic content was determined by the Folin-Ciocalteau’s spectrophotometric method. The thrombolytic activity was assessed by measuring clot lysis ability whereas the membrane stabilizing activity was evaluated by heat- and hypotonic solution-induced hemolysis assay. Tail immersion procedure and acetic acid- induced writhing model were used to measure the analgesic activity of C. radicans. The hypoglycemic, anti-diarrheal and CNS antidepressant activities were determined by oral glucose tolerance test, castor oil-induced diarrheal model and thiopental-sodium induced sleeping time test in mice, respectively.ResultsAll the organic soluble fractions of C. radicans contained phenolic compounds varying from 6.38 to 60.13 mg of GAE/gm of extractive, while in DPPH assay, EASF showed the highest free radical scavenging activity with IC50 is 4.69 μg/ml. The PESF exhibited highest thrombolytic activity (57.14% clot lysis) and the DMSF showed maximum 53.95% inhibition of heat-induced hemolysis of human RBCs. In both tail immersion and acetic acid induced writhing models, the PESF, DMSF, EASF at the doses of 200 and 400 mg/kg body weight, induced a significant (P < 0.001) decrease in the painful sensation in mice. Substantial (P < 0.05) anti-hyperglycemic activity of test samples was found in mice loaded with glucose at the same doses mentioned earlier. Castor oil induced diarrheal test of the plant extractives has shown significant effect in comparison to control group. In CNS antidepressant activity assay, the test samples were able to reduce the duration of sleep in mice caused by thiopental administration.ConclusionAll these findings revealed that C. radicans possess significant antioxidant, thrombolytic, membrane stabilizing, analgesic, hypoglycemic, anti-diarrheal and CNS antidepressant activities.

Phytochemical composition of Ecballium elaterium extracts with antioxidant and anti-inflammatory activities: Comparison among leaves, flowers and fruits extracts

Ecballium elaterium (squirting cucumber) is a Mediterranean plant used in folk medicine. Especially fruits and fruits juice are administered for several therapeutic uses, although they can be toxic at high doses. In the present work, a phytochemical characterization of different parts of the plant, namely fruits, flowers and leaves, was made. Extracts were analyzed by liquid chromatography coupled to diode array detector and mass spectrometry with triple quadrupole analyzer (HPLC-DAD-MS/MS). Only one cucurbitacin, Cuc D, was found in the three extracts, and an additional one, Cuc P, was tentatively identified for the first time in fruits extract. Additionally, several flavonoids such as rutin, narcissin and kaempferol were tentatively identified in the plant. Antioxidant activity was evaluated by DPPH, ABTS, FRAP and inhibition of human erythrocyte hemolysis mediated by AAPH. Results revealed that flowers extract was the most active one, with an EC50 of 46.01 µg/ml for DPPH and TEAC values of 489.11 and 160.97 µmol Trolox/g extract for ABTS and FRAP respectively; despite the protective effect of all extracts in AAPH assay was relatively moderated. In case of anti-inflammatory activity, all extracts were active by inhibiting heat induced protein denaturation in a non-dose dependent manner, showed protection against hypotonic hemolysis and reduction of mice hind paw edema, 3 h after carrageenan injection; as for previous tests, flowers extract exhibited the highest percentage of inhibition (82.93%) of hind paw edema at a dose of 200 mg/kg. Therefore, E. Elaterium flowers extract showed potential antioxidant and anti-inflammatory effects similar to those exerted by the fruits.

Volume expansion of erythrocytes is not the only mechanism responsible for the protection by arginine-based surfactants against hypotonic hemolysis.

A novel arginine-based cationic surfactant Nα-benzoyl-arginine dodecylamide (Bz-Arg-NHC12) was synthesized in our laboratory. In this paper we study the interaction of Bz-Arg-NHC12 with sheep and human red blood cells (SRBC and HRBC respectively) due to their different membrane physicochemical/biophysical properties. SRBC demonstrated to be slightly more resistant than HRBC to the hemolytic effect of the surfactant, being the micellar structure responsible for the hemolytic effect in both cases. Moreover, besides the hemolytic effect, a dual behavior was observed for the surfactant studied: Bz-Arg-NHC12 was also able to protect red blood cells against hypotonic lysis for HRBC in a wide range of surfactant concentrations. However, the degree of protection showed for SRBC was about 50% lower than for HBRC. In this regard, a remarkable volume expansion was evidenced only for SRBC treated with Bz-Arg-NHC12, although no correlation with the antihemolytic potency (pAH) was found. On the contrary, our surfactant showed a greater pAH when human erythrocytes were submitted to hypotonic stress, with a low volume expansion, showing a higher amount of solubilized phospholipids in the supernatant when compared with SRBC behavior. Surface plasmon resonance measurements show the molecular interaction of the surfactant with lipid bilayers from HRBC and SRBC lipids, demonstrating that in the latter neither microvesicle release or lipid extraction occurred. Our results demonstrate that the volume expansion of erythrocytes is not the only mechanism responsible for the protection by surfactants against hypotonic hemolysis: volume expansion could be compensated via microvesicle release or by the extraction of membrane components upon collisions between red blood cells and surfactant aggregates depending on the membrane composition.

Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy.

The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells’ shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

Ex vivo encapsulation of dexamethasone sodium phosphate into human autologous erythrocytes using fully automated biomedical equipment

Erythrocyte-based drug delivery systems are emerging as potential new solutions for the release of drugs into the bloodstream. The aim of the present work was to assess the performance of a fully automated process (EDS) for the ex-vivo encapsulation of the pro-drug dexamethasone sodium phosphate (DSP) into autologous erythrocytes in compliance with regulatory requirements. The loading method was based on reversible hypotonic hemolysis, which allows the opening of transient pores in the cell membrane to be crossed by DSP. The efficiency of encapsulation and the biochemical and physiological characteristics of the processed erythrocytes were investigated in blood samples from 34 healthy donors. It was found that the processed erythrocytes maintained their fundamental properties and the encapsulation process was reproducible. The EDS under study showed greater loading efficiency and reduced variability compared to previous EDS versions. Notably, these results were confirmed using blood samples from Ataxia Telangiectasia (AT) patients, 9.33±1.40 and 19.41±2.10mg of DSP (mean±SD, n=134) by using 62.5 and 125mg DSP loading quantities, respectively. These results support the use of the new EDS version 3.2.0 to investigate the effect of erythrocyte-delivered dexamethasone in regulatory trials in patients with AT.

Biconcave shape of human red-blood-cell ghosts relies on density differences between the rim and dimple of the ghost's plasma membrane

The shape of the human red blood cell is known to be a biconcave disk. It is evident from a variety of theoretical work that known physical properties of the membrane, such as its bending energy and elasticity, can explain the red-blood-cell biconcave shape as well as other shapes that red blood cells assume. But these analyses do not provide information on the underlying molecular causes. This paper describes experiments that attempt to identify some of the underlying determinates of cell shape. To this end, red-blood-cell ghosts were made by hypotonic hemolysis and then reconstituted such that they were smooth spheres in hypo-osmotic solutions and smooth biconcave discs in iso-osmotic solutions. The spherical ghosts were centrifuged onto a coated coverslip upon which they adhered. When the attached spheres were changed to biconcave discs by flushing with an iso-osmotic solution, the ghosts were observed to be mainly oriented in a flat alignment on the coverslip. This was interpreted to mean that, during centrifugation, the spherical ghosts were oriented by a dense band in its equatorial plane, parallel to the centrifugal field. This appears to be evidence that the difference in the densities between the rim and the dimple regions of red blood cells and their ghosts may be responsible for their biconcave shape.

Breed and seasonal variations in erythrocyte osmotic fragility of goat kids raised in semi-arid savannah

The aim of this study was to evaluate the influence of sex, breed and season on erythrocyte osmotic fragility (EOF) as a biomarker of erythrocyte membrane integrity and oxidative stress in Red Sokoto (n = 60) and Sahel (n = 60) kids exposed to the peak of the three distinct seasons prevailing in the Northern Guinea Savannah Zone of Nigeria. The results revealed that irrespective of breed, thermal stress during the hot-dry season (HDS) had greater influence on EOF causing significant (P 0.05) sex difference in the EOF of both breeds. In conclusion, breed and season dramatically influence the susceptibility of erythrocytes of kids to hypotonic haemolysis with heat stress during the HDS causing decrease in membrane integrity in both breeds of goats. However, sexual immaturity may be responsible for lack of sexual dimorphism in EOF in both breeds. The use of mitigation techniques and prevention of additional stress factors such as weaning is recommended during the peak of the HDS.

Modified hydroxyethyl starch protects cells from oxidative damage

This article describes the synthesis of novel starch-antioxidant conjugates, which show great potential for biomedical applications to protect cells from oxidative damage. These conjugates were synthesized by the modification of a hydroxyethyl starch (molecular weight=200,000g/mol) with various sterically hindered phenols that differ in radical scavenging activity. They possess substantial radical scavenging activity toward a model free radical. It was found that the polymer conjugate conformation depends on the antioxidant structure and degree of substitution. We constructed the complete conformational phase behavior for the polymers with increasing degrees of substitution from small-angle neutron scattering data. It was observed that the conjugate conformation changes are the result of water shifting from a thermodynamically favorable solvent to an unfavorable one, a process that then leads to compaction of the conjugate. We selected the conjugates that possess high substitution degree but still exhibit coil conformation for biological studies. The high efficiency of the conjugates was confirmed by different in vitro (hypotonic hemolysis of erythrocytes/osmotic resistance of erythrocytes and the change of [Ca2+]i inside freshly isolated cardiomyocytes) and in vivo (acute hemorrhage/massive blood loss) methods.

Sinomenine and magnoflorine, major constituents of Sinomeni caulis et rhizoma, show potent protective effects against membrane damage induced by lysophosphatidylcholine in rat erythrocytes.

The effects of the water extract of Sinomeni Caulis et Rhizoma (SCR-WE) and its major constituents, sinomenine (SIN) and magnoflorine (MAG), on moderate hemolysis induced by lysophosphatidylcholine (LPC) were investigated in rat erythrocytes and compared with the anti-hemolytic effects of lidocaine (LID) and propranolol (PRO) as reference drugs. LPC caused hemolysis at concentrations above the critical micelle concentration (CMC), and the concentration of LPC producing moderate hemolysis (60%) was approximately 10μM. SCR-WE at 1ng/mL-100μg/mL significantly inhibited the hemolysis induced by LPC. SIN and MAG attenuated LPC-induced hemolysis in a concentration-dependent manner from very low to high concentrations (1 nM-100μM and 10 nM-100μM, respectively). In contrast, the inhibiting effects of LID and PRO on LPC-induced hemolysis were observed at higher concentrations (1-100μM) but not at lower concentrations (1-100 nM). Neither SIN nor MAG affected micelle formation of LPC, nor, at concentrations of 1 nM-1μM, did they attenuate the hemolysis induced by osmotic imbalance (hypotonic hemolysis). Similarly, SCR-WE also did not modify micelle formation or hypotonic hemolysis, except at the highest concentration. These results suggest that SIN and MAG potently protect the erythrocyte membrane from LPC-induced damage and contribute to the beneficial action of SCR-WE. The protective effects of SIN and MAG are mediated by some mechanism other than prevention of micelle formation or protection of the erythrocyte membrane against osmotic imbalance.

Influence of sex, reproductive status and foetal number on erythrocyte osmotic fragility, haematological and physiologic parameters in goats during the hot-dry season

The current study was aimed at evaluating the effect of heat stress (during the hot-dry period) on some physiological variables, haematology and erythrocyte osmotic fragility (EOF) in bucks, pregnant (single and twin) and lactating Red Sokoto (RS) goats. Forty apparently healthy adult goats were used for the study and were allotted to four groups [bucks (n = 10), pregnant (n = 10) dry (n = 10), and lactating (n = 10) does]. The pregnant goats were further re-grouped according to number of foetuses (single foetus, n = 5 and twins, n = 5). The temperature-humidity index and physiological variables measured were significantly higher in the afternoon compared to morning hours. Pregnant does had higher respiratory and pulse rates than the dry does, but no significant change (P &amp;gt; 0.05) in rectal temperature was observed between groups. On the basis of sex, bucks had lower (P &amp;lt; 0.05) mean corpuscular volume (MCV) than other groups, in addition to having higher (P &amp;lt; 0.01) mean corpuscular haemoglobin (MCH) and total leucocyte counts than dry does. Pregnant does exhibited significantly lower (P &amp;lt; 0.05) packed cell volume, but significantly higher (P &amp;lt; 0.05) MCH and mean corpuscular haemoglobin concentrations (MCHC) than lactating does. Does with a single foetus had significantly lower (P &amp;lt; 0.05) MCV, but higher MCHC than does with twin pregnancies. Using EOF as a biomarker of oxidative stress, erythrocytes of bucks were significantly more resistant to hypotonic haemolysis than those of dry, pregnant and lactating does, with no significant difference in EOF between does of different groups. The erythrocytes of single and twin pregnancies showed similar haemolysis pattern. In conclusion, sex, lactation, and the number of foetuses carried by pregnant does significantly influences physiological and haematological variables in RS goats during the hot-dry season. Also, during heat stress, the changes in physiological variables seem to enhance favourable adaptation by preventing an increase in rectal temperature even in twin pregnancies. &amp;nbsp;

Erythrocyte membranes from slaughterhouse blood as potential drug vehicles: Isolation by gradual hypotonic hemolysis and biochemical and morphological characterization

The present study was aimed at investigating the effect of isolation process-gradual hypotonic hemolysis on chosen parameters of the erythrocyte membranes (ghosts) originating from bovine and porcine slaughterhouse blood. The estimation of the gradual hypotonic hemolysis as a drug loading procedure for the erythrocyte ghosts was performed as well.Based on the results derived from analysis of the osmotic properties of the erythrocytes, the gradual hemolysis was performed with high volume of erythrocytes and 35mM hypotonic sodium-phosphate/NaCl, enabling >90% of hemolysis for both types of erythrocytes. Detailed insight into ghosts’ morphology by field emission-scanning electron microscopy revealed a distortion from erythrocyte shape and an altered surface texture with increased bilayer curvature for both samples. Compared to erythrocytes, an average diameter of ghosts from both type of erythrocytes decreased for only about 10%. The reported unidispersity of the isolated ghosts is of great importance for their potential application as vehicles of active compounds. Gradual hemolysis did not lead to substantial loss of cholesterol and membrane/cytoskeleton proteins. This result indicated the ghosts’ possibility to mimic the chemical and structural anisotropic environment of in vivo cell membranes, which is of significance for drug diffusion and partition coefficients. Induced shift of phosphatidylserine to external surface of the ghosts demonstrated their potential application as vehicles for targeted drug delivery to cells of reticuloendothelial system. Ultra high-performance liquid chromatography and Fourier transform infrared spectroscopy revealed the presence of a drug model – dexamethasone-sodium phosphate, and its interaction with structural components in both types of erythrocyte ghosts.

Global Thrombosis Test (GTT) can detect major determinants of haemostasis including platelet reactivity, endogenous fibrinolytic and thrombin generating potential

Detection of both thrombosis and bleeding risk are essential in clinical cardiology. Thrombin generated by activated platelets and from the extrinsic coagulation pathway is the major determinant of thrombogenesis and hemostasis. Although novel oral anticoagulants further increase the bleeding risk of antiplatelet drugs, platelet function tests do not reliably predict hemorrhagic complications. It seems that in addition to platelet aggregation, true assessment of bleeding risks requires the measurement of both platelet and plasma derived thrombin activity. To adapt a novel, near-patient test for the assessment of both antithrombotic and anticoagulant effects of oral thrombin inhibitors. The point-of-care Global Thrombosis Test (GTT), which measures platelet reactivity to shear-activation in native blood, was used. Thrombin, generated from activated platelets (procoagulant activity) plays a pivotal role in GTT measurement. In order to assess endogenous thrombin potential, in a separate blood sample thrombin generation was induced by microparticles formed during hypotonic hemolysis. Thus two blood samples were tested to measure simultaneously platelet reactivity (occlusion time, OT) and hemolysis (microparticles)-induced endogenous thrombin potential (OT-H). In healthy subjects (n=32), OT measured in native blood was reduced in hemolysed blood (100% vs. 43 ± 4%; OT vs. OT-H respectively). Shortening of OT in hemolysed blood (OT-H) was dose-dependently inhibited by the in vitro added thrombin inhibitor argatroban. In patients receiving dabigatran (n=27), OT and, to a lesser extent, OT-H was prolonged, compared to healthy volunteers. Intra-assay variation of OT-H was low (4.5%), but interindividual variation was great, both in healthy subjects (61%) and in patients on dabigatran (65%). Thrombin inhibitors argatroban, heparin (in vitro) and dabigatran (in vivo) all prolonged both OT and OT-H. There was no correlation between the measured OT and OT-H data. Microparticles shed from erythrocytes during hypotonic lysis of native blood considerably shortened OT. In a direct proportion to the applied concentrations, various thrombin inhibitors prolonged both OT (antithrombotic effect) and to a lesser extent, OT-H (anticoagulant effect). Further large studies are required to evaluate the usefulness of this technique in a clinical setting, in assessing the anticoagulant and antithrombotic effects of medication and relating GTT results with observed thrombotic and bleeding events.