Hemoglobin function in the developing goat and sheep

Comparative studies of the blood of newly born goats and sheep have indicated a number of mechanisms which are responsible for a decreasing affinity for molecular oxygen in these developing animals during the first 40 to 60 days after birth.
The concentration of 2,3-DPG in the red cells of young goats increases four to sixfold during the first 3 to 4 days of life, and this increase is associated with a marked decrease in the cellular pH; 2,3-DPG does not bind to hemoglobins of goats and the decreased affinity for oxygen of goat blood at this period is apparently due to the lowered pH produced by the large increase of intracellular anions. Similar changes occur in young lambs.
After 15 to 20 days the changes of the dissociation curve are related more to structural differences between adult and fetal hemoglobins; cellular pH moves closer to the values of adult red cells. In goats of this age the predominate hemoglobins are those with β chains and these have dissociation curves shifted further to the right than other adult hemoglobins.
In young lambs Hb-C is found only in association with Hb-A but the amount present seldom exceeds 5 to 10%. The oxygen affinities of sheep Hb-A and Hb-C are identical but higher than that of sheep Hb-B.     

Electrophoretic characterization of the hemoglobins in pocket gophers of the genus Thomomys.

1. Hemoglobin phenotypes have been determined for closely related pocket gopher subspecies in the genus Thomomys from sea level and high altitude. 2. A single hemoglobin band was demonstrated for each gopher population with cellulose acetate membrane electrophoresis, with a mobility of 73% relative to human Hb-A. 3. Using polyacrylamide gel electrophoresis, a major Hb component (94% of the total Hb) showed a relative mobility of 68-69%; the minor Hb component showed a relative mobility of 74-75%. 4. The electrophoretic and physiological similarities of the gopher hemoglobins have persisted despite extreme differences in ambient PO2 in the habitats of each subspecies.     

Oxidative Activation of K-Cl Cotransport by Diamide in Erythrocytes from Humans with Red Cell Disorders,and from Several Other Mammalian Species

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0–1.0 mm) in K-free Cl or NO₃ media of variable osmolalities (200–450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO₃) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) ∼ rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0.00001) and in NO₃ (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity. Received: 5 June 1996/Revised: 4 October 1996     

Comparison of transfusion with DCLHb or pRBCs for treatment of intraoperative anemia in sheep.

Isoflurane-anesthetized sheep were transfused with packed red blood cells (pRBCs) or diaspirin cross-linked hemoglobin (DCLHb) for treatment of intraoperative hemorrhage. A rapid 15-min hemorrhage with lactated Ringer (LR) infusion maintained filling pressure at baseline and reduced blood hemoglobin (Hb) to ~5 g/dl. Sheep received 2 g/kg Hb, DCLHb (n = 6), or pRBCs (n = 7); control group received LR alone (n = 6). After 2 h, anesthesia was discontinued; sheep were monitored in the animal intensive care unit for 48 h. DCLHb expanded blood volume more, but increased total blood Hb less, than pRBCs. Lower Hb and increased methemoglobin resulted in lower arterial oxygen content compared with the pRBCs. DCLHb caused pulmonary hypertension (from 13 to 30 mmHg) and elevated filling pressure (from 6 to 15 mmHg). Cardiac outputs (CO) were similar for all groups during anesthesia; however, during recovery CO increased only in the LR and packed pRBCs groups. DCLHb may limit the reflex ability to increase CO after volume expansion. Hemodynamic effects of DCLHb may be exaggerated when infused after large-volume LR.     

Nuclear magnetic resonance and oxygen affinity study of cesium binding in human erythrocytes.

We investigated the interaction of the cesium ion (Cs(+)) with the anionic intracellular components of human red blood cells (RBCs); the components studied included 2,3-bisphosphoglycerate (BPG), ADP, ATP, inorganic phosphate (P(i)), carbonmonoxy hemoglobin (COHb), and RBC membranes. We used spin-lattice (T(1)) and spin-spin (T(2)) (133)Cs NMR relaxation measurements to probe Cs(+) binding, and we found that Cs(+) bound more strongly to binding sites in BPG and in RBC membranes than in any other intracellular component in RBCs at physiologic concentrations. By using James-Noggle plots, we obtained Cs(+) binding constants per binding site in BPG (66 +/- 8 M(-1)), ADP (19 +/- 1 M(-1)), ATP (25 +/- 3 M(-1)), and RBC membranes (55 +/- 2 M(-1)) from the observed T(1) values. We also studied the effect of Cs(+) on the oxygen (O(2)) affinity of purified Hb and of Hb in intact RBCs in the absence and in the presence of BPG. In the absence of BPG, the O(2) affinity of Hb decreased upon addition of Cs(+). However, in the presence of BPG, the O(2) affinity of Hb increased upon addition of Cs(+). The O(2) affinity of Cs(+)-loaded human RBCs was larger than that of Cs(+)-free cells at the same BPG level. (31)P NMR studies on the pH dependence of the interaction between BPG and Hb indicated that the presence of Cs(+) resulted in a smaller fraction of BPG available to bind to the cleft of deoxyHb. Our NMR and O(2) affinity data indicate that a strong binding site for Cs(+) in human RBCs is BPG. A partial mechanism for Cs(+) toxicity might arise from competition between Cs(+) and deoxyHb for BPG, thereby increasing oxygenation of Hb in RBCs, and thus decreasing the ability of RBCs to give up oxygen in tissues. The presence of Cs(+) at 12.5 mM in intact human RBCs containing BPG at normal concentrations did not, however, alter significantly the O(2) affinity of Hb, thus ruling out the possibility of Cs(+)-BPG interactions accounting for Cs(+) toxicity in this cell type.     

Tangential flow filtration of hemoglobin

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS‐PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P₅₀) and cooperativity coefficient (n) were regressed from the measured oxygen‐RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC‐MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α‐ and β‐ globin chains. Taken together, our results demonstrate that HPLC‐grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb‐based oxygen carriers. © 2008 American Institute of Chemical Engineers, 2009     

Membrane-bound hemoglobin as a marker of oxidative injury in adult and neonatal red blood cells

A comparative study of the effect of hydrogen peroxide on adult and neonatal red blood cell (RBC) membrane protein composition has been carried out. The results indicate that (a) the native neonatal RBC membranes contain higher levels of membrane-bound hemoglobin (MBHb) than the adult RBC membranes. (b) The content of MBHb increases when RBCs are incubated with increasing concentrations of hydrogen peroxide (H2O2), more so in neonatal than in adult RBCs; however, neonatal RBC membrane proteins are less susceptible to H2O2 oxidation than adult ones. This could be attributed to the fact that Hb F, which is more susceptible to oxidation than Hb A, adds to the reduction potential of neonatal RBC (in which it is present in large amounts) and partially protects neonatal membrane proteins against oxidant stress compared to Hb A in adult RBC. (c) In both neonatal and adult RBCs, Spectrin 1 is relatively more susceptible to oxidant stress than spectrin 2, and spectrins in adult RBC are more labile for peroxidation than the spectrins in neonatal RBC. (d) Based on electrophoretic studies with and without reduction of membranes with mercaptoethanol, we have classified two types of MBHb: Type I is adsorbed to membrane by noncovalent interactions and Type II MBHb is chemically crosslinked to membrane components by disulfide bridges; the content of both these types increases when RBCs are incubated with increasing concentrations of H2O2. (e) Band 6 protein is present in higher amounts in neonatal than in adult RBC membranes. (f) Since the total content of MBHb increases linearly with the level of oxidant stress, we suggest that it could be used as a marker for oxygen radical-induced injury to tissues.     

Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.     

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O(2) carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs.     

Preparation and in vivo evaluation of two bovine hemoglobin-based plasma expanders

A hemoglobin (Hb)-based oxygen carrier was successfully transfused into rats. An ultrapure lipid-free bovine Hb was prepared by hypotonic dialysis and ultrafiltration. The Hb was polymerized with glutaraldehyde and the P50 was 24.3 mm Hg. On the basis of immunological analysis, immuno-dot blot, the Hb preparations were not antigenic. A second transfusion produced no adverse immunological side effects. A right shift in P50 was obtained by further treatment of polymerized Hb with inositol hexaphosphate; however, this Hb preparation was unsuitable for transfusion as all animals died within a few minutes. A 30% exchange transfusion in rats with the polymerized bovine Hb resulted in a 100% survival of all animals. P50 values of treated animals were reduced by about 2 mm Hg for 14 days. The Hb product circulated for 14 days as determined by 51Cr labeling. Ultrapure bovine Hb has the potential to circulate and carry oxygen in rats and causes no immunological side effects.     

Hemoglobins, XXVIII. Phosphate-protein-interaction, gene expression and function: the genetic and allosteric control of the oxygen affinity of the fetal blood (author's transl)

This work describes possible molecular mechanisms concerning the control of oxygen affinity in fetal blood of mammalia. There is a genetic control of oxygen affinity through a fetal gene: at constant phosphate concentration (Hb less than P2-glycerate) in humans there is a hemoglobin with only five binding sites to 2,3-bisphosphoglycerate, resulting in an increased oxygen affinity. In several species (sheep, cattle, goat) with Met-Leu as the N-terminal group of the beta-chains, the 2,3-bisphosphoglycerate binding sites are deleted in positions beta 1 and beta 2, so that the regulation is phosphate-independent and thus providing a fetal hemoglobin with an increased oxygen affinity. The allosteric control is observed in pigs. In the postembryonal development "adult" hemoglobin with seven contacts (beta-chains) is demonstrated. The increased oxygen affinity is achieved here by a reduced biosynthesis of 2,3-bisphosphoglycerate (Hb greater than P2-glycerate) (Rapoport-Luebering-cycle). The functional control is discussed with respect to the ontogeny of the hemoglobins.     

Estimation of nitric oxide concentration in blood for different rates of generation. Evidence that intravascular nitric oxide levels are too low to exert physiological effects

Endothelium-derived nitric oxide (NO) is a potent vasodilator in the cardiovascular system. Several lines of experimental evidence suggest that NO or NO equivalents may also be generated in the blood. However, blood contains a large amount of hemoglobin (Hb) in red blood cells (RBCs). The RBC-encapsulated Hb can react very quickly with NO, which is only limited by the rate of NO diffusion into the RBCs. It is unclear what the possible NO concentration levels in blood are and how the NO diffusion coefficient (D) and the permeability (Pm) of RBC membrane to NO affect the level of NO concentration. In this study, a steady-state concentration experimental method combined with a spherical diffusion model are presented for determining D and Pm and examining the effect of NO generation rate (V0) and hematocrit (Hct) on NO concentration. It was determined that Pm is 4.5 +/- 1.5 cm/s and D is 3410 +/- 50 microm2/s at 37 degrees C. Simulations based on experimental parameters show that, when the rate of NO formation is as high as 100 nm/s, the maximal NO concentration in blood is below 0.012 nM at Pm = 4.5 cm/s and Hct = 45%. Thus, it is unlikely that NO is directly exported or generated from the RBC as an intravascular signaling molecule, because its concentration would be too low to exert a physiological role. Furthermore, our results suggest that, if RBCs export NO bioactivity, this would be through NO-derived species that can release or form NO rather than NO itself.     

Model mice for Presbyterian hemoglobinopathy (Asn(beta108)-->Lys) confer hemolytic anemia with altered oxygen affinity and instability of Hb

Hb Presbyterian is a variant hemoglobin that carries Lys at Asn-108 of beta-globin. This variant Lys(beta108) residue enhances the stability of Hb in the deoxy-state, conferring the low affinity for oxygen-binding in vitro. In the present study, we generated mutant mice carrying the Presbyterian mutation (Asn(beta108)-->Lys) at the beta-globin locus by a targeted knock-in strategy. Heterozygous mice showed the expression of Hb Presbyterian in 27.7% of total peripheral blood without any hematological abnormalities, which well mimicked human cases. On the other hand, homozygous mice exclusively expressed Hb Presbyterian in 100% of peripheral blood associated with hemolytic anemia, Heinz body formation, and splenomegaly. Hb Presbyterian showed instability in an in vitro precipitation assay. Erythrocytes from homozygous mice showed a shortened life span when transfused into wild-type mice, confirming that the knocked-in mutation of Lys(beta108) caused hemolysis in homozygous mice. This is the first report on the hemolytic anemia of unstable hemoglobin in an animal model. These results confirm the notion that the higher ratio of an unstable variant beta-globin chain in erythrocytes triggers the pathological precipitation and induces hemolysis in abnormal hemoglobinopathies.     

Red Blood Cells: Centerpiece in the Evolution of the Vertebrate Circulatory System

All vertebrates except cold-water ice fish transport oxygenvia hemoglobin packaged in red blood cells (RBCs). VertebrateRBCs vary in size by thirtyfold. Differences in RBC size havebeen known for over a century, but the functional significanceof RBC size remains unknown. One hypothesis is that large RBCsare a primitive character. Agnathans have larger RBCs than domammals. However, the largest RBCs are found in urodele amphibianswhich is inconsistent with the hypothesis that large RBCs areprimitive. Another possibility is that small RBCs increase bloodoxygen transport capacity. Blood hemoglobin concentration ([Hb])and mean RBC hemoglobin concentration (MCHC) increase from Agnathato birds and mammals. However, the changes in [Hb] and MCHCdo not parallel changes in RBC size. In addition, RBC size doesnot affect blood viscosity. Thus, there is no clear link betweenRBC size and oxygen transport capacity. We hypothesize thatRBC size attends changes in capillary diameter. This hypothesisis based on the following observations. First, RBC width averages25% larger than capillary diameter which insures cell deformationduring capillary flow. Functionally, RBC deformation minimizesdiffusion limitations to gas exchange. Second, smaller capillariesare associated with increased potential for diffusive gas exchange.However, smaller capillaries result in higher resistances toblood flow which requires higher blood pressures. We proposethat the large capillary diameters and large RBCs in urodelesreflect the evolutionary development of a pulmonary vascularsupply. The large capillaries reduced systemic vascular resistancesenabling a single ventricular heart to supply blood to two vascularcircuits, systemic and pulmonary, without developing high pressureson the pulmonary side. The large RBCs preserved diffusive gasexchange efficiency in the large capillaries.     

Hemoglobin autofluorescence as potential long-term glycemic marker in the rat animal model

Diabetes is a serious disease whose patients often require long-term care. Blood glucose and intermediate glycation product of glycated hemoglobin (HbA1c) are, at best, surrogate biomarkers of disease progression. There is indication that advanced glycation end products (AGEs) better reflect diabetic risks. In this study, we explored the use of red blood cells (RBCs) and lysed hemoglobin (Hb) autofluorescence (AF) as potential biomarkers of diabetic complication. AF spectra measured under 370 nm excitation reveals that both RBC and Hb fluorescence in the 420 to 600 nm region. At early time points following diabetic induction in rats, AF increase in lysed Hb is more dramatic compared to that of RBCs. Moreover, we found significance variance of Hb autofluorescence despite relatively constant HbA1c levels. Furthermore, we found that although a correlation exists between AGE autofluorescence and HbA1c levels, the lack of complete correspondence suggests that the rate of AGE production differs significantly among different rats. Our results suggest that with additional development, both RBC and Hb autofluorescence from lysed RBCs may be used act long-term glycemic markers for diabetic complications in patients.      

Evidence for significant quantities of creatine kinase MM isoenzyme in human brain

Oxygen equilibrium studies have been carried out on hemoglobins A2 (alpha2delta2), Lepore-Washington (alpha2(deltabeta)2) and P-Nilotic (alpha2(beta2delta)2) using the beta chain containing hemoglobins A and S as controls. This investigation was initiated mainly because of controversial data that have been published on the oxygen affinity of hemoglobin (Hb) A2 and because samples containing the rare Hb P-Nilotic became available. Each hemoglobin was isolated in pure form by anion exchange chromatography; the samples used in the equilibrium analyses contained 100 mg Hb/dl with less than 5% ferrihemoglobin and no 2,3--diphosphoglycerate. Oxygen equilibrium analyses were made at 37 degrees C with the method of Benesch et al. (1965) Anal. Biochem. 11, 81--87; Anal. Biochem. 55, 245--248 (1973). A slight, but definite increase in oxygen affinity was observed for Hb A2 as well as for Hb P-Nilotic while the increase for the Hb Lepore-Washington was somewhat greater. The values for n, the Hill coefficient, and the Bohr effects were the same for all hemoglobin types. The differences in oxygen affinity of these hemoglobins apparently result from the differences in primary structure that are characteristic for those proteins.     

Erythrocytes possess an intrinsic barrier to nitric oxide consumption

It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K(Hb) approximately 10(7) M(-1) s(-1)) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500-1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant, K(RBC), would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K(RBC) approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500-1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs.     

In Vivo Volume and Hemoglobin Dynamics of Human Red Blood Cells

Human red blood cells (RBCs) lose ∼30% of their volume and ∼20% of their hemoglobin (Hb) content during their ∼100-day lifespan in the bloodstream. These observations are well-documented, but the mechanisms for these volume and hemoglobin loss events are not clear. RBCs shed hemoglobin-containing vesicles during their life in the circulation, and this process is thought to dominate the changes in the RBC physical characteristics occurring during maturation. We combine theory with single-cell measurements to investigate the impact of vesiculation on the reduction in volume, Hb mass, and membrane. We show that vesicle shedding alone is sufficient to explain membrane losses but not volume or Hb losses. We use dry mass measurements of human RBCs to validate the models and to propose that additional unknown mechanisms control volume and Hb reduction and are responsible for ∼90% of the observed reduction. RBC population characteristics are used in the clinic to monitor and diagnose a wide range of conditions including malnutrition, inflammation, and cancer. Quantitative characterization of cellular maturation processes may help in the early detection of clinical conditions where maturation patterns are altered.     

The Effects of Ethanol on the Morphological and Biochemical Properties of Individual Human Red Blood Cells

Here, we report the results of a study on the effects of ethanol exposure on human red blood cells (RBCs) using quantitative phase imaging techniques at the level of individual cells. Three-dimensional refractive index tomograms and dynamic membrane fluctuations of RBCs were measured using common-path diffraction optical tomography, from which morphological (volume, surface area, and sphericity); biochemical (hemoglobin (Hb) concentration and Hb content); and biomechanical (membrane fluctuation) parameters were retrieved at various concentrations of ethanol. RBCs exposed to the ethanol concentration of 0.1 and 0.3% v/v exhibited cell sphericities higher than those of normal cells. However, mean surface area and sphericity of RBCs in a lethal alcoholic condition (0.5% v/v) are not statistically different with those of healthy RBCs. Meanwhile, significant decreases of Hb content and concentration in RBC cytoplasm at the lethal condition were observed. Furthermore, dynamic fluctuation of RBC membranes increased significantly upon ethanol treatments, indicating ethanol-induced membrane fluidization.