In vitro kinetics of oxygen transport in erythrocyte suspension or unmodified hemoglobin solution from human and other animals

Oxygen transport behavior in erythrocyte suspension or in hemoglobin solution was studied as a potential therapeutic model for the clinical treatment of blood loss, and this can also provide physiological data with which to evaluate blood substitutes. In the present project, we examined the in vitro kinetics of hemoglobin binding to and releasing oxygen, to provide detailed oxygen-flux measurements for unmodified hemoglobin solutions and erythrocyte suspensions in human, as well as other vertebrates. An in vitro method was used, based on a widely used artificial system, with the oxygen saturation level being detected in real time. Results from this study indicated that the kinetic curves of human erythrocyte suspensions and hemoglobin solutions were either S-shaped or hyperbolic, respectively. Based on these curves, the significance of T(50) emerged in our investigation, where T(50) is defined as the time needed for 50% hemoglobin to be saturated with oxygen, and reflects the efficiency with which hemoglobin carries oxygen. This parameter may be used to diagnose blood diseases, and could be a standard for evaluating blood substitutes. In this study, we also compared the T(50) of 4 species of vertebrates, and found that it shows a distinct efficiency of oxygen binding related to species, and potentially reveals the evolutionary function of hemoglobin and its possible adaptation to the environment.     

Effect of nitrite-induced methemoglobinemia on the kinetics of blood deoxygenation

Kinetics of blood deoxygenation was studied during acute hypoxia induced by subcutaneous administration of sodium nitrite using polarographic method. Plasma oxygen tension remained unaltered as the dose of sodium nitrite increased, while the dynamics of oxygen release was dose-dependent. The constant of oxyhemoglobin deoxygenation rate proved to vary with blood deoxygenation. The nitrite-induced deceleration of oxyhemoglobin deoxygenation was due to the inactivation of a fraction of hemoglobin as well as to the increased hemoglobin oxygen affinity and possible changes in the oxygen permeability of erythrocyte membranes during acute methemoglobinemia.     

Kinetics of reaction of nitrite with deoxy hemoglobin after rapid deoxygenation or predeoxygenation by dithionite measured in solution and bound to the cytoplasmic domain of band 3 (SLC4A1)

The reaction of deoxyhemoglobin with nitrite was characterized in the presence of dithionite using hemoglobin in solution or bound to the cytoplasmic domain of band 3 (CDB3). Deoxyhemoglobin was generated by predeoxygenation (nitrogen flushing followed by addition of dithionite), or transiently, by rapidly mixing oxyhemoglobin with nitrite and dithionite simultaneously. Wavelength-dependent kinetic studies confirmed the formation of nitrosyl hemoglobin. Furthermore, the rate of reaction was independent of dithionite concentration, indicating that dithionite does not reduce nitrite to nitric oxide directly. Model simulation studies showed that superoxide anion generated by dithionite reduction of molecular oxygen was not a factor in the reaction kinetics. CDB3-bound hemoglobin reacted faster with nitrite than did hemoglobin in solution. This difference was most pronounced for predeoxygenated hemoglobin and least pronounced for rapidly deoxygenated hemoglobin. The smaller difference observed in the rapid deoxygenation experiment was associated with much faster kinetics compared to the predeoxygenation experiment. Model simulation studies showed, and literature evidence indicates, that faster kinetics in the rapid deoxygenation experiment were related to the initial presence of R-state Hb(II)O 2 alphabeta dimers, both in dilute solution and when bound to CDB3. Thus, rapidly deoxygenated CDB3-bound hemoglobin alphabeta dimers react 5-fold faster with nitrite than predeoxygenated tetrameric hemoglobin in solution. Faster nitrite reductase kinetics for CDB3-bound hemoglobin suggests the possibility of preferential nitric oxide generation at the inner surface of the erythrocyte membrane, thus coupling the release of oxygen from hemoglobin to the production and successful release of nitric oxide from the erythrocyte, and the regulation of blood flow.     

Nonequilibrium-Facilitated Oxygen Transport in Hemoglobin Solution

We have used the quasi-linearization method to obtain numerical solutions to the equations which describe steady-state diffusion of oxygen through layers of hemoglobin solution. The numerical solutions show how the facilitated flux of oxygen depends upon the layer thickness, reaction-rate coefficients, and other parameters of the system. The results indicate that steady-state oxygen diffusion in layers of hemoglobin solution, similar to those studied by Scholander, should be adequately described by the models which assume chemical equilibrium exists throughout the layer, but for layers of concentrated hemoglobin solution about the thickness of a human erythrocyte, the facilitation of oxygen diffusion should be much less than the equilibrium models predict.     

A reaction cell for simultaneous measurements of fluorescence and absorption in a hemoglobin solution at various oxygen pressures

An apparatus was constructed to carry out measurements of fluorescence, optical absorption and oxygen partial pressure in a hemoglobin or other solution simultaneously, and its performance was examined. This apparatus has a rhombiform optical cell in place of the usual square optical cell used in commercially available spectrofluorometers. Fluorescence emitted at the region near the cell surface in the solution could be detected satisfactorily and easily even if the solution had strong light absorption bands at both the excitation and the emission wavelengths in the presence of high concentrations of a chromophore. This apparatus was particularly effective for studies on the interactions of a fluorescent allosteric effector with hemoglobin at various degrees of deoxygenation. Consequently, it was proved experimentally that the fluorescence of β-naphthyl triphosphate bound to hemoglobin is completely quenched. Moreover, simultaneous and continuous measurements of the oxygen-binding equilibrium of hemoglobin and the allosteric effector-binding to hemoglobin as a function of oxygen partial pressure could be satisfactorily carried out, and it is confirmed that β-naphthyl triphosphate binds not only to deoxyhemoglobin but also to fully oxygenated hemoglobin and lowers strongly the oxygen affinity of hemoglobin as an allosteric effector.     

Current status of erythrocyte substitutes.

During the last two decades the search for alternatives to whole blood transfusions has led to promising developments in the field of erythrocyte substitutes. Hemoglobin solutions free of fragments of erythrocyte stroma and fluorocarbon emulsions are not blood-type-specific and appear likely to satisfy some proportion of our blood requirements. Both must be modified before becoming clinically useful. The oxygen affinity of the hemoglobin solution must be reduced and its intravascular persistence improved. Fluorocarbons cannot yet contribute significantly to the oxygen supply unless the patient breathes hyperbaric oxygen. Recent advances are leading to solutions for these problems.     

The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties

The process of oxygen-dependent hemoglobin induction in Daphnia magna was studied over an 11-day period of hypoxia (ambient oxygen partial pressure: 3 kPa). Along with the increase of hemoglobin concentration in the hemolymph, hemoglobin became the dominant protein fraction in gel filtration experiments using extracts of whole animals. The size of the native aggregates was constant. However, subunit composition depended on the duration of hypoxia: the pattern of predominantly expressed subunits under hypoxia deviated from that of normoxic individuals. The varying degree of hypoxic induction for different hemoglobin subunits was confirmed by autoradiography. Along with changes in hemoglobin subunit composition, oxygen affinity of the respiratory protein increased. The dynamics of the hemoglobin induction process was analysed. Newly synthesized hemoglobin can be detected within 18 h after the onset of hypoxia. A marked increase in hemoglobin concentration is evident from the third day of hypoxia, and a steady state of hemoglobin concentration is reached within 11 days. The changes of hemoglobin subunit expression in response to hypoxia form the structural basis for the observed adjustments of hemoglobin function leading to enhanced oxygen transport at low ambient oxygen concentrations.     

Oxygen binding to sickle cell hemoglobin.

The extent of oxygen binding and light scattering of concentrated solutions of hemoglobin S have been determined as a function of oxygen partial pressure using a thin film optical cell. Nearly reversible oxygen binding is observed as witnessed by the small hysteresis found between slow deoxygenation and reoxygenation runs. High co-operativity is noted from unusually large concentration-dependent Hill coefficients when aggregated hemoglobin S is present. The application of linkage theory with the inclusion of non-ideal solution properties permits a test of various simple models for oxygen binding to both the monomer (α₂β₂^s) and polymer (aggregated) phase. It is concluded that oxygen binding to the polymer is either negligible or small under present experimental conditions. Phase diagrams of the solution concentration in equilibrium with polymer phase as a function of oxygen partial pressure are derived using best fit values of polymer parameters.     

Visible spectroscopic technique for flowing erythrocytes in capillary

An optical spectroscopic system for determining the rate of oxygen release from flowing erythrocytes in microvessel is developed. The apparatus consists of following units attached to an inverted microscope. 1) A scanning spectrophotometer, equipped with a grating and a photon counter, was connected to an eyepiece of the microscope through a narrow light-guide, as to obtain the absorption spectrum (wave length range: 450-650 nm) of a focused spot (phi = 7 microns). 2) The velocity of erythrocyte flow was measured by dual-spots cross-correlation method, using two photomultipliers (connected to A/D converter and microcomputer) with two light-guides inserted into another eyepiece. 3) The diameter of vessel was estimated from digitized video-images, using a color image-processor. The ability of the apparatus was tested with (a) hemoglobin solution, (b) flowing erythrocyte suspension and (c) capillaries of rat mesentery. The rate of oxygen release through the vessel wall was calculated.     

Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

Binding of organic phosphates by human hemoglobin at alkaline pH-values

The hemoglobin-oxygen equilibrium binding curve was found to be sensitive to the addition of inositol hexaphosphate at pH 9.1. A solution of hemoglobin A in 0.050 $\text{M}$ sodium borate was half-saturated with oxygen at a partial pressure of 0.55mm Hg. Hemoglobin A in 0.050 $\text{M}$ sodium borate, 0.001 $\text{M}$ inositol hexaphosphate, pH 9.1 was half-saturated with oxygen at a partial pressure of 0.95mm Hg. The Hill plot was linear with a slope of 2.0 in the absence of phosphates. In the presence of inositol hexaphosphate the slope of the Hill plot increased from 1.0 to 2.36. The dependence of fractional saturation of hemoglobin with oxygen on concentration of inositol hexaphosphate was determined at partial pressures of oxygen of 0.46 and 1.07mm Hg.     

Interaction of hemoglobin with the red blood cell membrane. A saturation transfer electron paramagnetic resonance study

Human hemoglobin has been labeled on cysteine 93(beta) with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and reassociated with erythrocyte membrane previously stripped of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase. The affinity of hemoglobin for the membrane is not affected by the presence of the label. Saturation transfer electron paramagnetic resonance measurements show that the diffusion rotational movements of hemoglobin are considerably slowed down when it is bound to the erythrocyte membrane. The correlation time of rotation, tau c, is found to be 8 . 10(-6) s as compared with 2 . 10(-8) s when the hemoglobin molecule is in solution. The same values are obtained whether the protein is associated with its high- or low-affinity binding sites. They depend on the viscosity of the solution. The high-affinity sites are presumably located on the segment of the band 3 protein which extends into the cytoplasm and which links through ankyrin, the spectrin-actin cytoskeleton to the membrane. When band 3 is cross-linked into a dimer after reaction with the copper-ortho-phenanthroline chelate, the correlation time of rotation of spin-labelled hemoglobin is unchanged. It is also independent of the presence of the spectrin-actin network and ankyrin. These results show tha the movements of hemoglobin bound by ionic linkage to different part (protein or phospholipid) of the cytoplasmic surface of the membrane are similarly highly restricted by some potential or energetic barrier. They give also evidence for independent movements and flexibility in the assembly of the macromolecules which link the spectrin-actin cytoskeleton to the erythrocyte membrane.     

Oxygen supply to isolated rabbit heart during its perfusion with eryhem and blood

Coronary vessels of the isolated rabbit heart were perfused with eryhem--a solution containing 2.5--3.0 g% of hemoglobin and with the rabbit own blood diluted to the Hb content corresponding to such in eryhem. The oxygen capacity of eryhem as per 1 g of Hb constituted 72% only of the oxygen capacity of erythrocyte Hb. It was shown that eryhem was capable of uptake, transportation and of giving up oxygen. At the same time the myocardium perfused with eryhem was in worse conditions of oxygen supply than the muscle perfused with blood. The required level of oxygen uptake was reached with the increase of coronary blood supply and more complete utilization of blood oxygen reserves.     

Heritable components of feline hematology, clinical chemistry, and acid-base profiles

Four erythrocyte variables (erythrocyte count, hemoglobin, mean cell volume, packed cell volume), 14 serum variables (alanine transferase, albumin, alkaline phosphatase, calcium, chloride, cholesterol, creatinine, glucose, phosphorus, potassium, sodium, total protein, triglycerides, urea nitrogen), and 7 venous acid-base variables (base excess, bicarbonate, carbon dioxide partial pressure, oxygen partial pressure, oxygen saturation, pH, and total carbon dioxide) were evaluated for heritability in domestic cats (Felis catus). Values used for individual cats were expressed as the mean over all lifetime measurements, using 444-530 animals for clinical chemistry, 629 animals for acid-base, and 564 animals for erythrocyte metrics. Gender and age at death (where applicable) also were evaluated for correlation with variables. Heritabilities for clinical chemistry, acid-base, and erythrocyte variables ranged, respectively, from 0.13 to 0.78, from 0.23 to 0.59, and from 0.41 to 0.69 (P < 0.05). This result indicates that serum variability has a genetic basis and is segregating in this feline population. These findings may have important implications in both research and clinical medicine.     

Oxygen equilibrium of emulsified solutions of normal and sickle hemoglobin.

Emulsions containing microdroplets of concentrated solutions of normal or sickle hemoglobin dispersed in a continous oil phase have been prepared, and the aggregation of sickle hemoglobin within microdroplets in a deoxygenated emulsion demonstrated. The equilibrium oxygen saturation of hemoglobin in the emulsions has been measured as a function of the partial pressure of oxygen by a novel spectrophotometric technique which corrects for the scattering of light in the emulsion. The half-saturation oxygen pressure and cooperativity of oxygen binding are substantially greater in concentrated (27 g/dl) solutions of sickle hemoglobin than in solutions of non-aggregating hemoglobin. The shape of the oxygen equilibrium curve of concentrated sickle hemoglobin is qualitatively discussed in terms of a previously proposed model (1).     

The Hypoxic Stress on Erythrocytes Associated with Superoxide Formation

Super oxide is produced during the authorization of hemoglobin. Authorization of hemoglobin is, however, facilitated under hypoxic conditions where hemoglobin is only partially oxygenated.

We have recently found that the erythrocyte superoxide dismutase does not fully react with the additional superoxide produced under hypoxic conditions. A leakage of superoxide from the erythrocyte is thus detected, resulting in a potential source for oxyradical damage to tissues.

Detailed studies on intact erythrocytes as a function of oxygen pressure have now been performed. These studies further delineate the hypoxic stress on erythrocytes and the mechanism for the leakage of superoxide. By centrifugation of samples under various oxygen pressures it was possible to show an enhanced rate of lysis at reduced oxygen pressures with a maximum rate in the region of 25 mm Hg. At much lower pressures where the hemoglobin is mostly deoxygenated the rate of lysis was dramatically decreased with almost no lysis detected even after three days. Lysis is shown to be associated with superoxide membrane damage. The formation of superoxide which does not react with endogenous SOD reaches a maximum value at much lower pressures where most of the hemoglobin is deoxygenated. It is suggested that the leakage at low pressure is associated with the formation of superoxide by oxidation of hemoglobin associated with the membrane.     

Adenine phosphoribosyltransferase: A simple spectrophotometric assay and the incidence of mutation in the normal population

The significance of partial deficiency of erythrocyte adenine phosphoribosyltransferase (APRT), reported in a number of subjects with gout, has been investigated by studying its incidence in 700 normal blood donors. Three clearly deficient subjects were found, an incidence not significantly different from that in patients with abnormalities of urate metabolism. A new assay method for APRT is described in which an erythrocyte lysate is incubated with adenine and phosphoribosylpyrophosphate (PRPP) for a given time; both hemoglobin and adenine nucleotide (AMP) are then precipitated with lanthanum phosphate; the change in absorbance of adenine at 260 nm reflects the extent of its conversion to AMP by APRT.This work was supported by the National Health and Medical Research Council of Australia.     

Studying the influence of some reactive oxygen species on physical and chemical parameters of blood

The aim of this work was to estimate the dynamics of blood physical and chemical parameters when blood specimens were processed by singlet oxygen in vitro. Our experiments were executed with whole blood specimens of healthy people (n = 10). Each specimen was divided into five separate portions of 5 mL. The first portion was a control (without any exposures). The second one was processed by an oxygen-ozone mixture (at ozone concentration of 500 μg/L, the third portion by oxygen, and the fourth and fifth ones were processed by a gas mixture with singlet oxygen (50 and 100% of generator power). In blood samples after processing we studied the activity of lactate dehydrogenase, aldehyde dehydrogenase and superoxide dismutase, erythrocyte and plasma levels of glucose and lactate, acid-base balance and the partial pressure of gases in blood. It was found out, that blood processing by singlet oxygen leads to optimization of energy, detoxication and antioxidant enzymes functioning with changes in plasma and erythrocyte level of glucose and lactate, normalization of blood gases level and acid-base balance. Our results show, that the effect of singlet oxygen on enzyme activity is more pronounced than exposure to an oxygen-ozone gas mixture.     

Redistribution of intestinal microcirculatory oxygenation during acute hemodilution in pigs.

Acute normovolemic hemodilution (ANH) compromizes intestinal microcirculatory oxygenation; however, the underlying mechanisms are incompletely understood. We hypothesized that contributors herein include redistribution of oxygen away from the intestines and shunting of oxygen within the intestines. The latter may be due to the impaired ability of erythrocytes to off-load oxygen within the microcirculation, thus yielding low tissue/plasma Po(2) but elevated microcirculatory hemoglobin oxygen (HbO(2)) saturations. Alternatively, oxygen shunting may also be due to reduced erythrocyte deformability, hindering the ability of erythrocytes to enter capillaries. Anesthetized pigs underwent ANH (20, 40, 60, and 90 ml/kg hydroxyethyl starch; ANH group: n = 10; controls: n = 5). We measured systemic and mesenteric perfusion. Microvascular intestinal oxygenation was measured independently by remission spectrophotometry [microcirculatory HbO(2) saturation (muHbO(2))] and palladium-porphyrin phosphorescence quenching [microcirculatory oxygen pressure in plasma/tissue (muPo(2))]. Microcirculatory oxygen shunting was assessed as the disparity between mucosal and mesenteric venous HbO(2) saturation (HbO(2)-gap). Erythrocyte deformability was measured as shear stress-induced cell elongation (LORCA difractometer). ANH reduced hemoglobin concentration from 8.1 to 2.2 g/dl. Relative mesenteric perfusion decreased (decreased mesenteric/systemic perfusion fraction). A paralleled reduction occurred in mucosal muHbO(2) (68 +/- 2 to 41 +/- 3%) and muPo(2) (28 +/- 1 to 17 +/- 1 Torr). Thus the proposed constellation indicative for oxygen off-load deficits (sustained muHbO(2) at decreased muPo(2)) did not develop. A twofold increase in the HbO(2)-gap indicated increasing intestinal microcirculatory oxygen shunting. Significant impairment in erythrocyte deformability developed during ANH. We conclude that reduced intestinal oxygenation during ANH is, in addition to redistribution of oxygen delivery away from the intestines, associated with oxygen shunting within the intestines. This shunting appears to be not primarily caused by oxygen off-load deficit but rather by oxygen/erythrocytes bypassing capillaries, wherein a potential contributor is impaired erythrocyte deformability.     

A Computational Model of a Microfluidic Device to Measure the Dynamics of Oxygen-Dependent ATP Release from Erythrocytes

Erythrocytes are proposed to be involved in blood flow regulation through both shear- and oxygen-dependent mechanisms for the release of adenosine triphosphate (ATP), a potent vasodilator. In a recent study, the dynamics of shear-dependent ATP release from erythrocytes was measured using a microfluidic device with a constriction in the channel to increase shear stress. The brief period of increased shear stress resulted in ATP release within 25 to 75 milliseconds downstream of the constriction. The long-term goal of our research is to apply a similar approach to determine the dynamics of oxygen-dependent ATP release. In the place of the constriction, an oxygen permeable membrane would be used to decrease the hemoglobin oxygen saturation of erythrocytes flowing through the channel. This paper describes the first stage in achieving that goal, the development of a computational model of the proposed experimental system to determine the feasibility of altering oxygen saturation rapidly enough to measure ATP release dynamics. The computational model was constructed based on hemodynamics, molecular transport of oxygen and ATP, kinetics of luciferin/luciferase reaction for reporting ATP concentrations, light absorption by hemoglobin, and sensor characteristics. A linear model of oxygen saturation-dependent ATP release with variable time delay was used in this study. The computational results demonstrate that a microfluidic device with a 100 µm deep channel will cause a rapid decrease in oxygen saturation over the oxygen permeable membrane that yields a measurable light intensity profile for a change in rate of ATP release from erythrocytes on a timescale as short as 25 milliseconds. The simulation also demonstrates that the complex dynamics of ATP release from erythrocytes combined with the consumption by luciferin/luciferase in a flowing system results in light intensity values that do not simply correlate with ATP concentrations. A computational model is required for proper interpretation of experimental data.