Molecular sieving of red cell membranes during gradual osmotic hemolysis

Summary Rat red blood corpuscles were held stationary with respect to a continuously flowing solution in either a specially constructed centrifuge or in glass filters. The concentration of the solution was gradually decreased to cause the swelling and subsequent gradual osmotic hemolysis of the cells. The passage of the intracellular molecules —potassium, adenylate kinase, and hemoglobin—across the cell membranes and into the flowing solution was determined as a function of time. Ions and molecules begin passage across the membranes in the order of increasing molecular size. The initial flow of potassium is followed by the initial flows of hemoglobin and adenylate kinase. The flow of hemoglobin has been interpreted as the flows of hemoglobin monomers, dimers, and tetramers such that the time sequence is: potassium; hemoglobin monomer; adenylate kinase/hemoglobin dimer; and finally, hemoglobin tetramer. It is concluded that the stressed cell membrane has molecular sieving properties and that the exclusion limit (effective hole size) increases as a function of time during the initial stages of gradual osmotic hemolysis. The process of gradual osmotic hemolysis is discussed in terms of molecular sieving through stress-induced effective membrane holes. It is suggested that a portion of the membrane protein might form an elastic network which would account for the gradual increase in size and apparent homogeneity of the effective holes.This work was prepared under the auspices of the U.S. Atomic Energy Commission.     

The use of ELISA to monitor amplified hemolysis by the combined action of osmotic stress and radiation: potential applications

A new assay has been developed to study the osmotic fragility of red blood cells (RBCs) and the involvement of oxygen-derived free radicals and other oxidant species in causing human red blood cell hemolysis. The amount of hemoglobin released into the supernatant, which is a measure of human red blood cell hemolysis, is monitored using an ELISA reader. This ELISA-based osmotic fragility test compared well with the established osmotic fragility test, with the added advantage of significantly reduced time and the requirement of only 60 mul of blood. This small amount of blood was collected fresh by finger puncture and was immediately diluted 50 times with PBS, thus eliminating the use of anticoagulants and the subsequent washings. Since exposure of RBCs to 400 Gy gamma radiation caused less than 5% hemolysis 24 h after irradiation, the RBC hemolysis induced by gamma radiation was amplified by irradiating the cell in hypotonic saline. The method was validated by examining the protective effect of Trolox, an analog of vitamin E and reduced glutathione (GSH), a well-known radioprotector, against human RBC hemolysis caused by the combined action of radiation and osmotic stress. Trolox, a known membrane stabilizer and an antioxidant, and GSH offered significant protection. This new method, which is simple and requires significantly less time and fewer RBCs, may offer the ability to study the effects of antioxidants and membrane stabilizers on human red blood cell hemolysis induced by radiation and oxidative stress and assess the osmotic fragility of erythrocytes.     

Footstrike is the major cause of hemolysis during running.

There is a wide body of literature reporting red cell hemolysis as occurring after various forms of exercise. Whereas the trauma associated with footstrike is thought to be the major cause of hemolysis after running, its significance compared with hemolysis that results from other circulatory stresses on the red blood cell has not been thoroughly addressed. To investigate the significance of footstrike, we measured the degree of hemolysis after 1 h of running. To control for the potential effects of oxidative and circulatory stresses on the red blood cell, the same subjects cycled for 1 h at equivalent oxygen uptake. Our subjects were 10 male triathletes, who each completed two separate 1-h sessions of running and cycling at 75% peak oxygen uptake, which were performed in random order 1 wk apart. Plasma free hemoglobin and serum haptoglobin concentrations were measured as indicators of hemolysis. We also measured methemoglobin as a percentage of total hemoglobin immediately postexercise as an indicator of red cell oxidative stress. Plasma free hemoglobin increased after both running (P < 0.01) and cycling (P < 0.01), but the increase was fourfold greater after running (P < 0.01). This was reflected by a significant fall in haptoglobin 1 h after the running trials, whereas no significant changes occurred after cycling at any sample point. Methemoglobin increased twofold after both running and cycling (P < 0.01), with no significant differences between modes of exercise. The present data indicate that, whereas general circulatory trauma to the red blood cells associated with 1 h of exercise at 75% maximal oxygen uptake may result in some exercise-induced hemolysis, footstrike is the major contributor to hemolysis during running.     

Hypotonic hemolysis of human red blood cells: a two-phase process.

Previous use of hemolysis time measurement to determine permeability coefficients for the red blood cell membrane rested on the assumption that cells swelling in a hypotonic medium hemolyzed immediately on reaching critical volume. By preswelling red cells to various volumes prior to immersion in hemolytic solutions we extrapolate to the hemolysis time of red cells immersed at critical volume and thereby find a significant period of time during which the cells apparently remain in a spherical form prior to release of hemoglobin. Revised estimates of permeability coefficients follow from including this spherical (nonswelling) phase. In addition, the appreciation of a characteristic time period during which the membrane is under tension provides new opportunity to study physical and chemical properties of the membrane.     

Effects of low doses of gamma rays on the stability of normal and diabetic erythrocytes

We studied the influence of low doses of γ radiation (from 0.04 to 1.8 mGy) on the stability of human red blood cells (RBC) from healthy donors and diabetic patients using absorption spectroscopy. Because of the alteration of many enzymatic pathways in diabetic RBCs resulting in strong modification of the lipid and protein membrane components one could expect that the ionizing γ-radiation should influence the stability of the healthy and diabetic cells in a different way. Indeed, distinct discontinuities and monotonic changes of hemolysis detected in the healthy and diabetic RBCs suggest that various enzymatic and chemical processes are activated in these membranes by γ radiation. M?ssbauer measurements showed that only the highest applied dose of γ radiation caused modification of hemoglobin in both types of RBCs.     

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Hypotonic hemolysis of human red blood cells: a two-phase process

Summary Previous use of hemolysis time measurement to determine permeability coefficients for the red blood cell membrane rested on the assumption that cells swelling in a hypotonic medium hemolyzed immediately on reaching critical volume. By preswelling red cells to various volumes prior to immersion in hemolytic solutions we extrapolate to the hemolysis time of red cells immersed at critical volume and thereby find a significant period of time during which the cells apparently remain in a spherical form prior to release of hemoglobin. Revised estimates of permeability coefficients follow from including this spherical (nonswelling) phase. In addition, the appreciation of a characteristic time period during which the membrane is under tension provides new opportunity to study physical and chemical properties of the membrane.Presented in part at the 1974 joint meeting of the Biophysical Society and the American Society of Biological Chemists.     

Electrical hemolysis of human and bovine red blood cells

Summary The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50% hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50% hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

Release of hemoglobin from the erythrocyte during intravascular hemolysis contributes to the pathology of a variety of diseased states. This effect is partially due to the enhanced ability of cell-free plasma hemoglobin, which is primarily found in the ferrous, oxygenated state, to scavenge nitric oxide. Oxidation of the cell-free hemoglobin to methemoglobin, which does not effectively scavenge nitric oxide, using inhaled nitric oxide has been shown to be effective in limiting pulmonary and systemic vasoconstriction. However, the ferric heme species may be reduced back to ferrous hemoglobin in plasma and has the potential to drive injurious redox chemistry. We propose that compounds that selectively convert cell-free hemoglobin to ferric, and ideally iron-nitrosylated heme species that do not actively scavenge nitric oxide, would effectively treat intravascular hemolysis. We show here that nitroxyl generated by Angeli's salt (sodium alpha-oxyhyponitrite, Na2N2O3) preferentially reacts with cell-free hemoglobin compared to that encapsulated in the red blood cell under physiologically relevant conditions. Nitroxyl oxidizes oxygenated ferrous hemoglobin to methemoglobin and can convert the methemoglobin to a more stable, less toxic species, iron-nitrosyl hemoglobin. These results support the notion that Angeli's salt or a similar compound could be used to effectively treat conditions associated with intravascular hemolysis.     

Inhibition of the heme-induced hemolysis of red blood cells by the chlorite-based drug WF10

Excessive release of hemoglobin from red blood cells markedly disturbs the health status of patients due to cytotoxic effects of free hemoglobin and heme. The latter component is able to initiate novel hemolytic events in unperturbed red blood cells. We modeled this process by incubation of ferric protoporphyrin IX with freshly isolated red blood cells from healthy volunteers. The heme-induced hemolysis was inhibited in a concentration-dependent manner by the chlorite-based drug WF10, whereby the hemolysis degree was totally abolished at a molar ratio of 1:2 between chlorite and heme. Upon incubation of heme with WF10, the ultraviolet-visible spectrum changed, whereas the release of iron from heme and the appearance of fluorescent breakdown products of the porphyrin ring were negligible at this ratio, but increased with increasing excess of chlorite over heme. Thus, inhibition of hemolysis by WF10 takes already place at those chlorite concentrations, where no degradation of the porphyrin ring occurs. As WF10 is applied in form of an intravenous infusion to patients with severe inflammatory states, these data support the hypothesis that the beneficial WF10 effects are closely associated with inactivation of free heme.     

Developmental changes in human erythrocyte carbonic anhydrase levels: coordinate expression with adult hemoglobin

In order to bolster the argument that parallel developmental changes in erythrocyte adult hemoglobin (HbA) and carbonic anhydrase (CA) content provide a potentially suitable model for the dissection of coordinate gene expression, the magnitude of fetal vs adult differences in CA I and CA II levels was examined in human red cell subpopulations obtained after varying periods of exposure to CA-dependent, NH4Cl-HCO-3-mediated, acetazolamide-modulated hemolysis. When content of CA I and CA II was immunologically assessed in cohorts surviving successively longer periods of hemolysis, cord blood red cells were divisible into two populations. Fifteen to thirty percent are rapidly disrupted and have CA I and CA II concentrations similar to those in adult blood erythrocytes. The remaining 70 to 85% have CA I concentrations which are 100-fold less and CA II concentrations which are 5- to 20-fold less than those found in adults. Thus, contrary to past reports, the magnitude of the developmental change in CA I concentration closely resembles the magnitude of change in HbA levels.     

Transitory postnatal hemolysis of calf red cells by amino acids

Summary Among the amino acids which can be solubilized to give a concentration of 300 mM at near physiological pH, histidine and proline caused a complete hemolysis of newborn calf but not of adult cow red cells within 20 to 30 minutes at 38°C. While hydroxyproline, valine, and serine resulted in a partial lysis of calf cells, threonine, glutamine, and glycine were inefective. In this communication, emphasis has been focused on the mode of the lytic process by histidine, which was found to be affected by several governing parameters including the pH, temperature and the extracellular salts in the solution. Unlike human red cells suspended in isotonic histidine, both calf and cow cells lost little Na and K ions. In the presence of 300 mM histidine, both calf and cow cells displayed an instantaneous uptake of histidine amounting to 20 to 45 moles/ml RBC followed by a slow influx rate of 0.25 to 0.5 moles/ml RBC×min. The extent to which histidine entry was allowed by the cell was counterbalanced by Cl^– efflux, resulting in little change in cell volume prior to hemolysis. Moreover, histidine-induced hemolysis can by prevented by 1 mM or lower PCMBS without at discernible effect on histidine influx suggesting a possible membrane lesion or damage at the outer surface of the cell.Hemolysis induced by histidine decreased substantially when a calf reached two months of age which time the red cells containing the fetal hemoglobin are virtually depleted. The results of hemoglobin electroiphoresis obtained during this postnatal period revealed that those cells resistant to histidine hemolysis almost invariably contain the adult type hemoglobin suggesting a selective, speicific action of the amin acid on the featal cells.A preliminary report of these data has been presented at the 19th Annual Meeting (1975) of the Biophysical Society, Philadelphia, Pennsylvania.     

A novel device for the non-invasive measurement of free hemoglobin in blood bags.

The production of red blood cell concentrates from human donors is a very expensive procedure and human resources are in short supply. Under perfect storage conditions at a temperature of 2-6 degrees C, a blood bag must be used within 35-49 days (in Germany). Visual inspection of the bag for apparent hemolysis by a blood bank physician is a crucial but subjective quality control assessment. Since an interruption of the cold chain cannot be definitely ruled out, bags are often disposed of prematurely for safety reasons. There is currently no method of testing a closed blood bag with respect to hemolysis for its suitability to be used in a transfusion. The proposed optical measuring device is a hemoglobin sensor which determines the free hemoglobin in standard erythrocyte concentrates without opening the bag. The optical measurements are done on the flexible tube connected to the main bag. The optical measurements were evaluated using standard hemoglobin solutions with an accuracy of 0.005 g/dL. These investigations show that in the future each blood bag can be tested non-invasively for its content of free hemoglobin. This will contribute to decreasing the wastage rate of red blood cell concentrates.     

The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation

Nitric oxide (NO) plays a fundamental role in maintaining normal vasomotor tone. Recent data implicate a critical function for hemoglobin and the erythrocyte in regulating the activity of NO in the vascular compartment. Intravascular hemolysis releases hemoglobin from the red blood cell into plasma (cell-free plasma hemoglobin), which is then able to scavenge endothelium-derived NO 600-fold faster than erythrocytic hemoglobin, thereby disrupting NO homeostasis. This may lead to vasoconstriction, decreased blood flow, platelet activation, increased endothelin-1 expression (ET-1), and end-organ injury, thus suggesting a novel mechanism of disease for hereditary and acquired hemolytic conditions such as sickle cell disease and cardiopulmonary bypass. Furthermore, therapy with NO gas inhalation or infusion of sodium nitrite during hemolysis may attenuate this disruption in vasomotor balance by oxidizing plasma cell-free hemoglobin, thereby preventing the consumption of endogenous NO and the associated pathophysiological changes. In addition to providing an NO scavenging role in the physiological regulation of NO-dependent vasodilation, hemoglobin and the erythrocyte may deliver NO as the hemoglobin deoxygenates. While this process has previously been ascribed to S-nitrosated hemoglobin, recent data from our laboratories suggest that deoxygenated hemoglobin reduces nitrite to NO and vasodilates the human circulation along the physiological oxygen gradient. This newly described role of hemoglobin as a nitrite reductase is discussed in the context of blood flow regulation, oxygen sensing, and nitrite-based therapeutics.     

新型快速显微多道分光光度系统及其应用

介绍一种新型快速显微多道分光光度系统。利用倒置显微镜、衍射光栅、线阵CCD等构成的显微多道分光光度系统,可对微区样品在350nm-800nm（波长分辨率为0．2nm)光谱波长范围内进行快速光谱检测,最快检测时间为1ms。利用该系统进行的人血红细胞内血红蛋白测量分析表明,该具有高灵敏度、快速、无扰、在位、多光谱显微测量的特点,在生命科学研究中动态监测细胞生命过程的变化。     

Factors influencing variable oxidative hemolysis of inbred mouse erythrocytes

The hemolysis of erythrocytes from certain inbred mouse strains (e.g., BALB/c) in response to hydrogen peroxide stress has been shown to be correlated with the type of hemoglobin beta chain (Kruckeberg, W.C., et al. (1987) Blood 70, 909-914). The characteristic hemolytic response of BALB/c red cells to oxidative stress resembles that of human red cells in that carbon monoxide and iron chelators inhibit hemolysis of both. Gross hemoglobin oxidation rates were similar in hemolytic (BALB/c) and nonhemolytic (C57BL/6) strains. The rate and degree of in vitro catalase inhibition by sodium azide was also the same for the two strains. Even in the presence of this catalase inhibitor the assayable hydrogen peroxide disappeared within seconds of its addition, yet hemolysis was not observed for about 15 min. The mechanism underlying this delay between hydrogen peroxide addition and disappearance and subsequent hemolysis is under investigation.     

Differences between cation-osmotic hemolysis and filterability in exaprolol- and glutaraldehyde-treated human red blood cells

The changes in human red blood cell microrheology in different glutaraldehyde (3.0 and 5.0 x 10(-6) mol x l(-1)) and exaprolol (2.5 and 5.0 x 10(-4) mol x l(-1)) concentrations were studied. The method of millipore filtration was compared with the method of cation-osmotic hemolysis. Both drugs prolonged the filtration time. Cation-osmotic hemolysis in glutaraldehyde-treated cells was significantly lower in comparison with the control group. On the other hand, there was a significant increase in cation-osmotic hemolysis in exaprolol-treated cells. Besides cation-osmotic hemolysis and filterability of erythrocytes, we evaluated the medium cell volume (MCV) and the medium cell hemoglobin concentration (MCHC). No changes in MCV and MCHC in glutaraldehyde-treated cells were observed. However, the MCV was significantly lower and the MCHC was significantly higher in exaprolol-treated cells. In conclusion, we suggest that the method of cation-osmotic hemolysis is more sensitive than the filtration method for determination of red blood cell microrheology.     

Characteristics of the interaction of a treponemal hemolysin with rabbit erythrocytes

The mechanism of action on rabbit red cells of Treponema hyodysenteriae hemolysin was studied using volume analysis and release of hemoglobin. While fixation of the hemolysin on the erythrocytes is temperature independent, it appears that hemolysis is temperature dependent. The kinetics of hemolysis proceed according to a sigmoid curve characterized by a prelytic lag. The duration of the prelytic lag varies inversely with the quantity of hemolysin but the rate and the maximum value of hemolysis are directly proportional to the quantity of hemolysin. The effect of sucrose and trypan blue on the hemolysin and the red cells suggest that erythrocyte lysis is likely to be induced by the hemolysin in a way different from that known for other hemolytic agents.     

Reactions of Hemoglobiniferous Cells to Aced and Basic Dyes under Varying Conditions of H-Ion Activity

1. An attempt has been made to apply Loeb's concept of the amphoteric nature of proteins for the discrimination of suspected hemoglobiniferous substances from known hemoglobiniferous substances according to their reactions to acid and basic dyes with reference to the isoelectric point of hemoglobin (pH 6.8).

2. The substances in the cytoplasm of known hemoglobiniferous cells (red blood corpuscles, normoblasts and erythroblasts) of the lymph nodes, spleen and bone marrow of the albino rat, when suspended in buffered dye-sucrose solutions, retain eosin on the acid side of pH 7.0, but the substances of the Russell bodies, suspected of being hemoglobiniferous, do not retain eosin at all; and the cytoplasm of the plasma cells, also alleged to be slightly hemoglobiniferous, only retains eosin on the acid side of pH 6.4.

3. The only basic dye used which did not precipitate in buffer solutions was methylene blue. This did not react with hemoglobin in accordance with Loeb's concept, because it did not penetrate mature red blood corpuscles and in those immature erythrocytes which it did penetrate it was precipitated by the reticulum.

4. Therefore, from the results obtained with the acid dye, it is tentatively concluded that the substance in the Russell bodies and in the cytoplasm of the plasma cells are not hemoglobiniferous because they do not react as do the substances in known hemoglobiniferous cells with reference to the isoelectric point of hemoglobin.

5. More investigation, however, must be carried out on both fresh and fixed material before a final unequivocal answer can be made to this problem.