Washed Erythrocytes Research Articles

Ionophore A23187 reduces energy charge by enhanced ion pumping in suspended human erythrocytes.

Washed human erythrocytes were basically incubated in phosphate-buffered saline at 37 degrees C with different concentrations (1-5 mumol l-1) of the divalent cationophore A23187. This ionophore induced a decrease by about 75% of ATP content and energy charge (EC) and a concomitant increase in ADP and AMP contents in a dose-dependent fashion such that an inverse ATP/ADP relationship developed. EGTA at 1 mumol l-1 annihilated the effect of A23187 on energy status. When glucose was added to the basic incubation medium A23187 inclusion resulted in an elevated lactate production concomitantly with a partial restoration of EC. Introduction of Mg2+ to basic incubation medium containing glucose and the ionophore resulted in a sharp increase in lactate production with an energy state that was maintained at a control level. It is concluded that the low EC of erythrocytes obtained by this ionophore is the result of runaway ATPases dissociated from glycolysis. Since human erythrocytes are devoid of organelles other than the plasma membrane it is concluded that the ionophoric effect is exerted in the plasma membrane.

Bile salt- and lysophosphatidylcholine-induced membrane damage in human erythrocytes.

The interaction of bile salts and lysophosphatidylcholine (LPC) with membranes has implications both in understanding the aetiology of a number of gastrointestinal disorders, including gastritis, gastric ulcers and colonic cancer, and in enhancing drug absorption by various epithelia. The membrane toxicity of nine bile salts (the sodium (S) salts of chenodeoxycholate (CDC), deoxycholate (DC) and cholate (C) and their glycine (G) and taurine (T) conjugates) and LPC was determined using erythrocyte haemolysis as a model parameter. Washed human erythrocytes were incubated for 15-60 min at 20 degrees C with media buffered at pH 8, 7 and 6. Bile salt toxicity was shown to be a function of type, concentration, pH and contact time with the membrane. At pH 7 toxicity decreased in the order LPC greater than unconjugated dihydroxy salts (SDC and SCDC) greater than conjugated deoxycholates (SGDC and STDC) greater than conjugated chenodeoxycholates (SGCDC and STCDC) greater than unconjugated trihydroxy salt (SC) greater than conjugated trihydroxy salts (SGC and STC). Incubation with equimolar combinations of bile salts (SDC+SCDC; STCDC+SGDC; SDC+STDC) indicated that the resultant damage was an additive function of the damage induced by the individual bile salts.

Differential effects of calcium ions and calcium phosphate on cytotoxicity of bile acids

Unconjugated secondary bile acids can promote colon cancer by damaging colonic mucosa and consequently increasing epithelial proliferation. It has been proposed that dietary calcium inactivates intestinal bile acids either by a Ca2(+)-dependent precipitation or by binding to insoluble calcium phosphate (CaPi). We studied the molecular mechanisms of these opposing hypotheses by using hemolysis of erythrocytes as a model parameter for cytotoxicity. Washed human erythrocytes were incubated for 15 min with buffered media (pH 7.4) containing increasing amounts of different bile acids. Deconjugation and 7 alpha-dehydroxylation of mixtures of glycine- or taurine-conjugated cholate and chenodeoxycholate drastically increased their cytotoxicity. Parallel measurements, using a fluorescent micellar probe, indicated that micellar aggregation is a prerequisite for this bile acid-induced lysis. Ca2+ concentrations up to 15 mM did not precipitate bile acids but stimulated cytotoxicity of both deoxycholate (DC) and its glycine conjugate (GDC). Cytotoxicity of the taurine conjugate (TDC) was stimulated to a much lesser extent. Increasing amounts of CaPi precipitated micellar DC and GDC, but not TDC, and consequently inhibited only cytotoxicity of the former two. These findings indicate that 1) hydrophobicity and micellar aggregation are important determinants of bile acid-induced cytotoxicity that explain the high cytotoxic potential of secondary bile acids in colon, and 2) cytotoxicity of bile acids is stimulated by free Ca2+ and inhibited by CaPi. This inhibition is due to binding of carboxylic (including secondary) bile acids to CaPi.

Laboratory Diagnosis of African Horse Sickness: Comparison of Serological Techniques and Evaluation of Storage Methods of Samples for Virus Isolation

Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA described in this paper was group specific. It did not require calibration with a standard positive serum but did yield elevated values with negative sera that were repeatedly frozen and thawed or heat inactivated. The IFA test was sensitive but could not be used on some field sera as the control cells exhibited fluorescence, possibly due to the animal being recently vaccinated with cell culture material. Sixty-two experimental sera were compared by VN, CF, AGID, and ELISA. Forty sera, 10 positive and 30 negative, were correctly classified by the 5 serologic assays. The 22 remaining sera gave mixed reactions. The AGID had no false positive results but had false negative results for up to 20% of the samples, depending upon the comparison. The VN, CF, and ELISA were similar in their variability. The length of time that virus could be recovered from a viremic blood sample was compared in an evaluation of storage methods for virus isolation samples. Washed erythrocytes were held at 4 C, washed erythrocytes plus stabilizer were held at -70 C, and blood that was drawn into a preservative (oxalate/phenol/glycerol) was held at 4 C.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of phenyldichloroarsine with erythrocytes.

The purpose of the study was to identify binding sites of organic arsenic in the erythrocyte and to explain species differences in binding. Washed erythrocytes were exposed to graded concentrations of [U-14C]phenyldichloroarsine (PDA) in phosphate-buffered saline containing 0.1% glucose and 0.1% bovine serum albumin. At low PDA concentrations, all cells bound the arsenical rapidly (within 10 min) and quantitatively. Human, pig, hamster, guinea pig, and mouse erythrocytes approached saturation at 0.02-0.3 mumol PDA/10(9) cells, depending on the species. Saturation points correlated well with each respective species' erythrocyte glutathione content. In contrast, rat erythrocytes showed no sign of saturation at PDA loads as high as 3.0 mumol/10(9) cells. Hemolysates of PDA-treated erythrocytes were subjected to Sephadex G-75 gel filtration chromatography. 14C from rat hemolysate was distributed between the hemoglobin and small molecular weight (glutathione-containing) fractions. In all other species, the 14C eluted almost exclusively with the glutathione-containing fractions. In equilibrium dialysis experiments, human hemoglobin did not bind PDA, whereas rat hemoglobin bound 2 PDA/mol with Kd approximately 5 microM. In conclusion, glutathione is the principal binding site of phenyldichloroarsine in erythrocytes. In most species, the arsenical does not bind to hemoglobin, even though it has free (titratable) sulfhydryls considerably in excess of the glutathione concentration. In rat erythrocytes, phenlydichloroarsine binds both to glutathione and to hemoglobin. Arsenical binding by rat hemoglobin is presumably due to the unique location of the extra titratable cysteine in that protein.

Ventricular function and fatty acid metabolism in neonatal piglet heart.

Studies in which subcellular systems were used suggest that neonatal myocardium has a sharply limited capacity to metabolize fatty acids. The relationship of these findings to the intact heart was tested on piglets, 8 h to 12 days of age. Left ventricular (LV) performance, O2 consumption (MVO2), and fatty acid (FA) uptake and oxidation were measured. Hearts were perfused at 70 cmH2O pressure with buffer containing 2% bovine serum albumin, insulin (100 microU/ml), 5 mM glucose, and 1.5 mM lactate. 14C-labeled palmitate was added (net FA, 0.5 mM). Washed erythrocytes were used to assure adequate O2 delivery. LV end-diastolic pressure (EDP) was controlled with a fluid-filled balloon. FA oxidation was estimated by measuring 14CO2 production. Hearts less than 24 h (group I, n = 6), those approximately 3 days (group II, n = 5), and those 6-12 days of age (group III, n = 10) were compared. Measurements at a low EDP (2-4 cmH2O) and at a higher EDP (7-9 cmH2O) were compared. At the low EDP, rates of FA oxidation for groups I-III averaged 30.0 +/- 3.0, 31.4 +/- 2.9, and 50.2 +/- 2.6 nmol.min-1.g-1, respectively. These values increased to 43.8 +/- 3.7, 42.6 +/- 2.5, and 63.8 +/- 4.0 nmol.min-1.g-1, respectively, at the higher EDP level (P less than 0.01 for each group). Thus within a few hours of birth, pig hearts are able to oxidize long-chain FA, and the rate of oxidation is linked to mechanical function. However, both the oxidation rate and the percentage of MVO2 accounted for by FA oxidation are greater in older hearts.

The circulating erythrocytes of rainbow trout ( Salmo gairdneri)

1. 1. Washed erythrocytes from rainbow trout maintained nonnoxically at near-optimum temperature (14°C) on a high nutritional plane and seasonally-neutral photoperiod (12L:12D) with minimum disturbance were separated by velocity sedimentation at unit gravity into 10 fractions. 2. 2. On the basis of constancy of mean cell hemoglobin content, 81.9% of circulating red cells were judged to be mature. 3. 3. What may have been postmature cells accounted for a further 7.5% of the total. The remainder, 10.6%, were presumed to be juvenile. 4. 4. The 11-membered hemoglobin isomorph system exhibited significant ontogenetic variations in the abundances of several minor, but no major isomorphs. 5. 5. Ultrastructural examination confirmed that cell elongation during maturation is correlated with development of the marginal band microtubule system. 6. 6. Hemoglobin formation appeared to be sequentially correlated with the appearance and complexity of the segregation apparatus, and the abundances of mitochondria and polysomes. 7. 7. The question of postmaturational hemoglobin synthesis was not resolved.

Effects of calcium antagonists on membrane fluidity in hypertension--an electron spin resonance study.

We examined alterations in membrane fluidity of hypertension and the effects of calcium antagonists on these changes by means of an electron spin resonance (ESR) and spin-label technique. Washed erythrocytes from spontaneously hypertensive rats (SHRs; aged 4 and 10-13 weeks) and from patients with essential hypertension, or cultured vascular smooth muscle cells from SHRs were examined and compared with those from normotensive controls. Electron spin resonance spectra for a fatty acid spin label-agent (5-nitroxy stearate) in the membranes were obtained. The values of hyperfine splitting and other parameters of the spectra were significantly higher in erythrocytes from hypertensive rats than in normotensive controls. Similar results were obtained in cultured vascular smooth muscles. These findings indicate that the membrane fluidity might be decreased in hypertension. When calcium was loaded to erythrocytes with Ca-ionophore A23187, the fluidity was decreased. The alternative degrees were greater in hypertensive rats than in controls, and these hypertensive changes were antagonized by diltiazem or verapamil. The results revealed that abnormalities of membrane fluidity in hypertension might be more prominent in the presence of calcium. Further, it is suggested that calcium antagonists could correct these membrane abnormalities in hypertension.

Alterations in calcium-handling of erythrocytes in spontaneously hypertensive rats. Calcium-induced changes in the osmotic fragility of erythrocytes in hypertension.

To investigate the sensitivity to calcium of erythrocytes in hypertension, changes in the osmotic fragility of erythrocytes following Ca-loading were observed. Washed erythrocytes were obtained from spontaneously hypertensive rats (SHR, Okamoto and Aoki) and age-matched normotensive Wistar Kyoto rats (WKY). Treatment of erythrocytes with Ca-ionophore A23187 and Ca in the medium caused a reduction in the osmotic fragility which correlated with the Ca-concentration. The degree of alteration in the osmotic fragility of erythrocytes was greater in SHR than in WKY. Oral administration of hydralazine to SHR significantly reduced the blood pressure. However, the alterations in the osmotic fragility of erythrocytes secondary to Ca-loading were not different between hydralazine-treated and untreated SHR. In the presence of a Ca-antagonist (verapamil or diltiazem) in the medium, the reduction of the osmotic fragility of erythrocytes caused by Ca-loading was inhibited, and the differences between SHR and WKY were abolished by Ca-antagonists. These results suggest that the greater changes in osmotic fragility of erythrocytes caused by Ca-loading in SHR could be due to a genetic abnormality of Ca-handling by the cell membranes, and that this abnormality might cause an increase in intracellular Ca, which contributes, in part, to the pathogenesis of hypertension.

Adaptation of coenzyme stimulation assays for the nutritional assessment of vitamins B1, B2 and B6 using the Cobas Bio centrifugal analyser.

Adaptation of coenzyme stimulation assays for the nutritional assessment of thiamine, riboflavin and pyridoxine on the Cobas Bio centrifugal analyser are described. Whole blood was collected into acid-citrate dextrose, which preserves the erythrocytes, prior to assay for several days. Washed erythrocytes stored at -70 degrees C and subsequently thawed, showed altered enzyme activities. The methods offer improved precision over existing procedures and take advantage of the high throughput capabilities of the instrumentation.

Paroxysmal Nocturnal Hemoglobinuria: Erythrocyte Acetylcholinesterase Deficit Analyzed by Immunoassay and Fluorescence-Activated Sorting

An immunodisplacement assay based on a specific, solid-phase monoclonal antibody was designed to measure acetylcholinesterase in tissue extracts. Sample enzyme content was determined from the competitive reduction of binding of a purified acetylcholinesterase standard, with a detection limit of 5 ng or less. Washed erythrocyte membranes from six normal subjects averaged 1.8 units of acetylcholinesterase activity and 0.45 microgram of acetylcholinesterase content per milligram of total protein. Enzyme activity and content in samples from three patients with paroxysmal nocturnal hemoglobinuria (PNH) were reduced approximately in parallel, by as much as 70%. The residual cholinesterase had almost the same homospecific activity as the normal enzyme and was bound with equivalent affinity by six different antibodies. Therefore, the cholinesterase defect was dominated by enzyme loss rather than by structural abnormalities affecting enzyme function. Fluorescence-activated sorting of antibody-labeled erythrocytes revealed a bimodal population distribution. Up to 66% of the PNH cells lacked cholinesterase, and the rest had a near-normal enzyme content. Inasmuch as enzyme-deficient cells represent the complement-sensitive population, cell sorting may help in assessing clinical status and, perhaps, in developing new therapeutic modalities for PNH.

Phosphorylation of erythrocyte membrane liberates calcium

Washed and permeabilized human erythrocyte ghosts were found to discharge calcium on treatment with ATP. Concomitantly, there was a decrease in phosphatidylinositol (PI) and an increase in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP 2). These results support the hypothesis that an inositide shuttle, PIágPIPágPIP 2, operates to maintain intracellular Ca 2+ levels. The cation is thought to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with participation of both PO and fatty acid ester CO groups. These cages are stabilized by inter-headgroup hydrogen bonding. When the inositol group is phosphorylated in position 4 and 5, inter-lipid hydrogen bonding is disrupted and the cage opens to release its Ca 2+.

Effects of aspirin, indomethacin, and sodium salicylate on human erythrocyte membranes as detected with electron spin resonance spectroscopy

Electron spin resonance spectroscopy of probed samples was used to determine the structural changes in human erythrocyte membranes prior to and at intervals following ingestion of either 10 grains acetylsalicylic acid, 10 grains sodium salicylate, or 50 mg indomethacin by both male and female subjects. Analysis of erythrocytes from female subjects indicated a time-dependent disordering of the membrane over the eight hour period following aspirin ingestion while the cells of male subjects showed a slight membrane ordering over the same time period. Erythrocytes drawn from females at the beginning of the menstrual cycle showed the greatest amount of membrane disordering at one hour following aspirin ingestion, but by eight hours, the membrane structure had returned to that of control. The time dependent disordering in membrane structure of cells from females in the middle of the menstrual cycle was biphasic. Ingestion of indomethacin induced only slight membrane changes in both male and female subjects over the times examined. Ingestion of sodium salicylate by either men or women did not induce significant changes in erythrocyte membrane order. Washed erythrocytes when mixed with salicylate, aspirin, or indomethacin were either identical to control cells or slightly more ordered. This study suggests that aspirin-induced alterations in membrane structure may depend upon steroid hormone levels.

Ultrastructural Kinetics of Electrically Induced Fusion of Human Erythrocytes

Conditions for electrical breakdown and subsequent fusion of biological membranes have been reported for a variety of eukaryotic cell systems. Large fusion yields may be obtained by first causing cells to form pearl chains parallel to the electric flux lines in a high frequency a.c. field by a process known as dielectrophoresis. The dielectrophoretic force affords close positioning of the cells and facilitates maintenance and expansion of intercellular membrane and cytoplasmic contiguity initiated by one or more sharp square pulses directed through the pearl chains. Reversible disruption of the cell membrane occurs when the compressive electrical force caused by the square pulse exceeds a critical value.This investigation focused on elucidating the ultrastructural kinetic mechanism(s) involved in the electrically-induced fusion of human erythrocytes. In this case, rapid freeze copper sample holders for freeze-fracture were incorporated as electrodes into a circuit capable of performing the type of protocol described above (Fig. 1). Washed human erythrocytes were suspended in 0.3 M sucrose containing 25.7 μm latex beads for electrode separation.

Fatty acid composition of human erythrocyte membranes by capillary gas chromatography—mass spectrometry

Capillary gas chromatographic and gas chromatographic—mass spectrometric methods were employed for profiling total fatty acid content of human erythrocyte membranes. The protocol was designed to efficiently separate, identify, and accurately quantify the fatty acid composition in human erythrocyte membranes. Washed erythrocyte “ghosts” were saponified in aqueous methanolic sodium hydroxide solution and methylated with boron trichloride and acid catalysis. Extracted total fatty acid methyl esters (FAMEs) were analyzed using a highly polar cyanopropylsiloxane SP 2560 fused-silica capillary column. Total run time was 55 min, and 45 FAMEs were tentatively identified by relative retention times compared to those of known FAMEs. Confirmation of identities by mass spectral structure elucidation revealed saturated, mono- and polyunsaturated, and branched-chain FAMEs. The presence of four fatty aldehydes was also confirmed as dimethyl acetal derivatives. Identification of cis/trans isomers was based on relative retentin times and characteristic profile of the cis/trans FAME standard. Quantification of FAMEs for normal subjects showed some variation in relative amounts, consistent with expectations based on literature reports on total or phospholipid FAMEs from human erythrocytes. Separation of individual components of fatty acid families (n-3), (n-6), and (n-9) is demonstrated. Losses in relative amounts of polyunsaturated fatty acids upon storing samples were also detectable by this rapid method.

Rainbow trout red cells in vitro

1. 1. Washed rainbow trout erythrocytes incubated at 14°C in Eagle's minimal essential medium and Cortland saline displayed sharp reductions m volume and water content, nucleoside triphosphate, K + and Cl −1 concentrations. Mg 2+ and, to a lesser extent, Na + concentrations increased. 2. 2. Cellular to medium Cl − ratios were indicative of membrane hyperpolarization. 3. 3. Morphological irregularities were also observed. 4. 4. Oxygen consumption and hemoglobin system organization were not grossly affected. 5. 5. Supplementation with pyruvate stabilized nucleoside triphosphate concentrations for at least 24 hr, and reduced rates of volume and compositional change to some extent. 6. 6. Addition of norepinephrine at physiologically realistic levels led to stabilization of Cl − content and reductions in Mg 2+ accumulation and water loss. 7. 7. Transient but modest increases in K + and Ca 2+ were coupled, under these circumstances, with some decrease in Na + concentration. 8. 8. Factors which may contribute to the dysfunctional status of these cells in vitro are discussed.

Resistance of Human Erythrocytes Containing Elevated Levels of Vitamin E to Radiation-Induced Hemolysis

Human erythrocytes were isolated from the blood of healthy donors and then incubated in the presence of suspensions of alpha-tocopherol for 30 min at 37 degrees C. Unabsorbed tocopherol was removed by centrifugation using several washes of isotonic phosphate-buffered saline. Washed erythrocytes were resuspended to 0.05%. Hct and exposed to hemolyzing doses of 60Co gamma radiation, and hemolysis was monitored continuously by light scattering at 700 nm in a recording spectrophotometer. The extent of hemolysis with time was sigmoid and data analysis was carried out on the time taken for 50% hemolysis to occur (t50%). The vitamin E content of erythrocytes was significantly elevated by the incubation procedure and resulted in the cells exhibiting a significantly increased resistance to hemolysis as reflected by the extended t50% values. Oral supplementation of 500 IU of vitamin E per day to eight normal human subjects for a period of 16 days also resulted in their washed erythrocytes exhibiting a significant increase in resistance to radiation-induced hemolysis. When comparing vitamin E incubated cells with control cells, both the dose-reducing factor (DRF) and the time for 50% hemolysis quotient (Qt50%) were observed to increase with increasing radiation dose.

Feeding of Psoroptes ovis (Acari: Psoroptidae) on cattle.

Washed bovine erythrocytes labeled in vitro with radioactive chromium were resuspended in physiological saline and injected into an 11-month-old Hereford heifer that was heavily infested with the common scabies (sheep scab) mite of cattle and sheep, Psoroptes ovis . After 24 h the mites were harvested and the radioactivity measured. The mites contained an average of 1 μl of blood per 100 mg of mites and had digested ca. 37% of the hemoglobin. The data show that P. ovis mites ingest erythrocytes during feeding on cattle but not to the extent that they do while feeding on rabbits as determined in an earlier study.

Ingestion of Rabbit Erythrocytes Containing 51Cr-Labeled Hemoglobin by Psoroptes Spp. (Acari: Psoroptidae) that Originated on Cattle, Mountain Sheep, or Rabbits1

Washed rabbit erythrocytes labeled with radioactive chromium in vitro were injected into 3 groups of 2 adult rabbits each. One group was heavily infested with the sheep scab mite, Psoroptes ovis ; the 2nd group was infested with Psoroptes mite from Mexican Mountain Sheep, and the 3rd group was infested with the ear canker mite, P. cuniculi . After 36 h, mites and scabs were harvested and the radioactivity determined. At time of collections, mites from all groups had an average of 8.9 ± 2.0 μl of blood per 100 mg of mites. More than 70% of the consumed hemoglobin had been digested by the mites. The data suggest that the ingestion of whole blood is not specific to a particular mite species but may be common to Psoroptes mites when feeding on rabbit hosts.

Increased adhesion of erythrocytes to endothelial cells in diabetes mellitus and its relation to vascular complications.

We studied the adhesion of erythrocytes from 30 diabetic patients and 25 controls to human endothelial cells. Washed erythrocytes were labeled with 51Cr and added to confluent endothelial cells cultured from umbilical veins. After incubation at 37 degrees C, the nonadherent erythrocytes were removed by sequential washings. The percentage of erythrocytes adhering to cultured endothelium after each wash was significantly higher when erythrocytes were from diabetics than when they were from controls (P less than 0.005). After the fifth wash, the mean adhesion ratio (percentage of adhering diabetic red cells: percentage of adhering control red cells) was 2.33 (range, 0.8 to 5.2). Increased adhesion was related to the extent of vascular complications in the diabetics, as assessed by a vascular score. With the same technique, fewer erythrocytes adhered to plastic and to cultured human fibroblasts than to endothelial cells, although the adhesion of the diabetic red cells to these surfaces was higher than that of the controls. These results suggest that in diabetes there is an intrinsic erythrocyte abnormality that is related to vascular disease.