Adaptation to Hypoxia by Increased HbO<Subscript>2</Subscript> Affinity and Decreased Red Cell ATP Concentration

THERE is a decrease in the O₂ affinity of mammalian haemoglobin (Hb) as the levels of 2,3-DPG or ATP are increased, which is explained by an allosteric effect on the HbO₂ binding^1,2. Similar observations on amphibians³ and fish⁴, which have molar ratios of ATP to Hb similar to those of DPG to Hb in mammals, suggest that red cell organic phosphates modulate Hb function in all vertebrates. The adaptation of mammals to various hypoxic stresses involves reduced HbO₂ affinity^5–9, the attendant increase in O₂ “unloading” capacity being mediated by an increase in the concentration of red cell 2,3-DPG. We have found the opposite response in hypoxic fish and suggest that an increased O₂ affinity results in increased O₂ transport for the fish.     

The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers

We compared rates of oxygen transport in an in vitro capillary system using red blood cells (RBCs) and cell-free hemoglobins. The axial PO(2) drop down the capillary was calculated using finite-element analysis. RBCs, unmodified hemoglobin (HbA(0)), cross-linked hemoglobin (alpha alpha-Hb) and hemoglobin conjugated to polyethylene-glycol (PEG-Hb) were evaluated. According to their fractional saturation curves, PEG-Hb showed the least desaturation down the capillary, which most closely matched the RBCs; HbA(0) and alpha alpha-Hb showed much greater desaturation. A lumped diffusion parameter, K*, was calculated based on the Fick diffusion equation with a term for facilitated diffusion. The overall rates of oxygen transfer are consistent with hemoglobin diffusion rates according to the Stokes-Einstein Law and with previously measured blood pressure responses in rats. This study provides a conceptual framework for the design of a 'blood substitute' based on mimicking O(2) transport by RBCs to prevent autoregulatory changes in blood flow and pressure.     

The effects of sickling on ion transport. I. Effect of sickling on potassium transport

The conversion of red cells of patients with sickle cell anemia (S-S) from biconcave disk to sickle shape by removal of oxygen was found to increase the fraction of medium trapped in cells packed by centrifugation from 0.036 (S.E. 0.003) to 0.106 (S.E. 0.004). The fraction of water in the cells (corrected for trapped medium) was not affected by this shape transformation. Cation transport, however, was changed profoundly. S-S cells incubated in N₂ rather than O₂ showed net K loss with acceleration of both influx and outflux. That this change in K transport was due to the process of sickling was indicated by (1) the persistence of the effect in the absence of plasma, (2) the absence of the effect in hypoxic S-S cells in which sickling was inhibited by alkali or carbon monoxide, (3) the reversal of the effect when sickling was reversed by exposure to O₂, and (4) the independence of the effect from such potentially important factors as age of the cell population. The acceleration of K transport by sickling is probably mediated by modification of the cell surface rather than the cell interior since concentrated sickle hemoglobin solutions in O₂ or N₂ did not show selective affinity for K. In molecular terms, the effect of sickling on K transport can be explained by presuming that the shape change (1) opens pathways for the free diffusion of K, and (2) accelerates K transport by a non-diffusion carrier process. The evidence for the former mechanism included (a) dependence of K influx into sickled cells on the concentration of K in the medium, and (b) increase in the total cation content of sickled cells with increasing pH. Observations suggestive of a carrier process included (a) the failure of sickled cell K concentration to become equal to external K concentration even after 48 hours, (b) the deviation of the flux ratio from that characteristic of diffusion, and (c) the dependence of K influx on glycolysis.     

Erythrocytic ATP release in the presence of modified cell-free hemoglobin

The red blood cell (RBC) has been proposed as an O₂ sensor through a direct link between the desaturation of intracellular hemoglobin (Hb) and ATP release, leading to vasodilation. We hypothesized that the addition of cell-free Hb to the extracellular space provides a supplementary O₂ source that reduces RBC desaturation and, consequently, ATP release. In this study, the saturation of RBC suspensions was lowered by additions of deoxygenated hemoglobin-based oxygen carrier (HBOC) and then assayed for extracellular ATP. When an acellular human Hb intramolecularly cross-linked between α subunits (ααHb, p50 = 33 mmHg) was added to the red cell suspension, ATP production was significantly less than that in the presence of a lower p50 HBOC (Hb cross-linked between β subunits, ββHb, p50 = 8 mmHg). These results provide a potential mechanism for the O₂ affinity of HBOCs to interfere with a vasodilatory signal.     

Kinetics of subunit dissociation of partially oxygenated hemoglobin determined by haptoglobin binding

Human haptoglobin 1-1 binds very rapidly to hemoglobin dimers but not to tetramers. We have studied the binding kinetics of partially oxygenated Hb A to haptoglobin 1-1. Under the oxygenation conditions used for the measurement of the K₁ of oxygenation ($\text{Hb O}{}_2\text{Hb}\text{≤ 1\%}$, pO₂ ≤ 0.5 mm Hg), the dissociation kinetics were found to be 50 times faster than that of deoxy Hb A. This result suggested that the binding of one molecule of oxygen to hemoglobin tetramer changed the quaternary structure of the intersubunit α₁β₂ contact surface.     

Kinetics of carbon monoxide and oxygen binding to hemoglobin in human red blood cell suspensions studied by laser flash photolysis

The reaction kinetics of the binding of CO and O₂ to hemoglobin (Hb) in human red blood cell (RBC) suspensions have been examined using a 300 ns dye laser to photodissociate HbCO or HbO₂. Fast (halftime1?0 μs) and slow (5?ms) processes were seen after photolysis. The results indicate that neither the rate constants nor the activation energies for the binding of CO to the fast reacting form of Hb in the RBC are significantly different from that measured in solution in spite of the different environments. Rate constants determined for O₂ binding in RBC were intermediate between rates observed for reaction with fast and slow reacting forms of Hb and probably consist of contributions from each. The slow recombination of CO and O₂ probably has contributions both from reaction with slow reacting forms of Hb and from ligand that had diffused away from the RBC after photolysis.     

Alterations of red cell membrane proteins and hemoglobin under natural and experimental oxidant stress

We compared on red cell membrane proteins and hemoglobin (Hb) the effects of (i) natural oxidant stress that has been suggested to occur in a variety of oxidative hemolytic anemias, and (ii) experimental stress induced by hydrogen peroxide. SDS-polyacrylamide gel electrophoresis was used for protein analysis. Under natural conditions (thalassemias, hemoglobinopathies with Hb unstability), a high molecular weight polymer (HMWP) and variable amounts of globin mono- and dimers became apparent. Furthermore, a major 12 kDa polypeptide, its dimer, and conspicuous spectrin degradation products in the band 2.2–2.6 region occurred in a patient carrying the highly unstable Hb Hammersmith. Under experimental conditions, incubation of erythrocyte ghosts with H₂O₂ in the presence of minimal concentration (25 μM) of Hb generated a HMWP at the expense of membrane proteins, mainly spectrin. Incubation of a diluted (200 μM) membrane-free hemolysate with H₂O₂ induced a HMWP, an array of globin oligomers and a 12 kDa polypeptide similar to that mentionned above. Therefore, the damage to the red cell membrane present in various oxidative hemolytic anemias, including polypeptide polymerisation and breakdown, can be produced by experimental oxidant stress. These observations support the view that the alterations described in the patients result directly from oxidative reactions. However, we did not observe in the patient the sharp breakdown of polyunsaturated fatty acids that was triggered in vitro by H₂O₂ in the presence of Hb acting as a catalyst. In most cases, oligo- and polymers were resistant to β-mercaptoethanol, and the chemical nature of the underlying cross-links is discussed. To our knowledge, the 12 kDa polypeptide, that we consider as arising from globin proteolysis, has never been reported under pathological conditions.     

Time-course of the polycythemic response in normoxic and hypoxic mice with high blood oxygen affinity induced by cyanate administration

The Hb-O₂ affinity and the erythropoietic response as a function of time were studied in mice treated with sodium cyanate for up to 2 months. Cyanate increased the Hb-O₂ affinity in normoxic mice more than in chronically hypoxic mice. The hemoglobin concentration rose as a function of time both in normoxic and hypoxic conditions but reached higher levels in hypoxia. After 42 days of study (21 days of hypoxia) hemoglobin reached maximum levels and thereafter showed a plateau in both cyanate and control animals. It is concluded that a chronic left-shifted oxygen dissociation curve does not avoid the development of hypoxic polycythemia in mice. Moreover, prolonged cyanate administration potentiates the crythropoietic response to chronic hypoxia. Since polycythemia is an index of tissue hypoxia, the results show that the high hemoglobin affinity did not prevent tissue hypoxia in low PO₂ conditions. Results showing beneficial effects of high hemoglobin oxygen affinity induced by cyanate based on acute hypoxic expositions should be cautiously interpreted with regard to their adaptive value in animals chronically exposed to natural or simulated hypoxia.Abbreviations Hb hemoglobin - NaOCN sodium cyanate - ODC oxygen dissociation curve - P ₅₀ PO₂ at which hemoglobin is half saturated with O₂     

A theoretical model for studying the rate of oxygenation of blood in pulmonary capillaries

A mathematical analysis of the process of gas exchange in the lung is presented taking into account the transport mechanisms of molecular diffusion, convection and facilitated diffusion of the species due to haemoglobin. Since the rate at which blood gets oxygenated in the pulmonary capillaries is very fast, it is difficult to set up an experimental study to determine the effects of various parameters on equilibration rate. The proposed study is aimed at determining the effects of various physiological parameters on equilibration rate in pathological conditions.Among the significant results are that 1. dissolved oxygen takes longer to achieve equilibration across the pulmonary membrane and carbon dioxide attains equilibration faster, 2. the equilibration length increases with increase in blood velocity, haemoglobin concentration, calibre of pulmonary capillaries and fall in alveolar PO₂, 3. the alveolar PCO₂ and forward and backward reaction rates of haemoglobin with CO₂ do not materially affect the equilibration rate or length. 4. At complete equilibration, by the end of the pulmonary capillary 92% of the total haemoglobin has combined with oxygen and 8% free pigment is left which is present as carbamino haemoglobin, met haemoglobin, carboxy haemoglobin etc.These results are of some importance for anaemic conditions, muscular exercise, meditation, altitude physiology, hypo-ventilation, hyperventilation, etc.Symbols H⁺ hydrogen ion - O₂ oxygen - CO₂ carbondioxide - HbO₂ oxyhaemoglobin - HbCO₂ carbaminohaemoglobin - PO₂ partial pressure of O₂ - PCO₂ partial pressure of CO₂ - PaO₂ O₂ tension in arterial blood - PaCO₂ CO₂ tension in arterial blood - k₁ forward rate constant for Eq. (1) - k₂ backward rate constant for Eq. (1) - m₁ forward rate constant for Eq. (2) - m₂ backward rate constant for Eq. (2) - k equilibration rate - a radius of the capillary - Q velocity of blood - L length of the capillary - D₀ diffusion coefficient of O₂ - D_c diffusion coefficient of CO₂ - D_H diffusion coefficient of Hb - H total haemoglobin concentration - A matrix - c₁ concentration of dissolved O₂ in blood - c₂ concentration of HbO₂ in blood - c₃ concentration of dissolved CO₂ in blood - c₄ concentration of HbCO₂ in blood - c₅ concentration of haemoglobin - c1alv concentration of O₂ in the alveolar region - c_3alv concentration of CO₂ in the alveolar region - c_iven concentration of the ith species in venous blood - c_iart concentrations of the ith species in arterial blood - F _is concentrations of the species in dimensionless form - J₀, I₀ Bessel's functions - P_alvO₂ tension of O₂ in alveolar region - P_alvCO₂ tension of CO₂ in alveolar region.     

Methemoglobin and the Activities of Catalase and Superoxide Dismutase in Nucleated Erythrocytes of Scorpaena porcus (Linnaeus, 1758) under Experimental Hypoxia (in vitro)

The effect of hypoxia on nucleated red blood cells of the black scorpionfish (Scorpaena porcus) was studied in vitro. Deep hypoxia (the oxygen concentration was less than 1 mg O₂ L^–1; the norm was 7–8 mg O₂ L^–1) led to the transition of a part of the hemoglobin molecules to the ferric state (methemoglobin). The maximum increase in the concentration of methemoglobin was 32%. The accumulation of methemoglobin in red blood cells was accompanied by an increase in the activity of catalase and superoxide dismutase and a decrease in the content of reactive oxygen species in the cytoplasm of cells. It was shown that the formation of methemoglobin did not cause damage to the cytoplasmic membranes of red blood cells. The percentage of red blood cell lysis in deoxygenated (less than 1.0 mg O₂ L^–1) suspensions quantitatively coincided with the control values.

     

Hemoglobin regulates the migration of glioma cells along poly(ε‐caprolactone)‐aligned nanofibers

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O₂)‐carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε‐caprolactone) (PCL)‐aligned fibers was studied in cell culture media supplemented with the O₂ storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O₂ tensions ranging from 1 to 19% O₂. Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL‐aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O₂, while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O₂ levels. The control data suggests that decreasing the O₂ tension in the incubator from 5 to 1% O₂ decreased aggregate dispersion on the PCL‐aligned fibers. However, the addition of bovine Hb at 5% O₂ significantly improved aggregate dispersion. At 19% O₂, Hb did not impact aggregate dispersion. Also at 1% O₂, aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb‐based O₂ carriers can be utilized to improve O₂ availability and the migration of glioma spheroids on nanofibers. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1214–1220, 2014     

Influence of substrate transport on the activity of immobilized Papaver somniferum cells

Summary Intraparticle diffusion resistance was studied for Papaver somniferum cells immobilized by Ca alginate gel. In callus tissue, these plant cells convert codeinone to codeine. First, the diffusion rates of substrates in the gel were measured, followed by investigation of the consumption rates of the substrates by free cells. The consumption rate of sucrose was zero order in relation to sucrose concentration, whereas that of codeinone was first order in relation to its concentration. The oxygen consumption rate obeyed Michaelis-Menten type kinetics with respect to dissolved oxygen concentration. Combining the reaction rates and diffusion rates allows calculation of the extent of the effect of diffusion limitation on the overall reaction rates. The analysis showed that the effectiveness factor for each substrate was about unity and that the influence of diffusion resistance was negligible. However, the oxygen concentration decreased considerably inside the particle, and this may affect the activity of the plant cell for repeated use over a long time period. Thus, deactivation proceeds due to the oxygen deficit although the temporal reaction rate is not affected.Abbreviations C _c cell concentration (g/l) - C _cod codeinone concentration (g/l) - c _{O 2} dissolved oxygen concentration (g/l) - K _m constant in Eq. (3) (g/l) - K _cod rate constant in Eq. (1) (l/g of cells per second) - k _suc rate constant in Eq. (2) (g sucrose/g of cells per second) - R radius of particles (mm) - r distance from the centre of the particle (mm) - r _cod consumption rate of codeinone (g codeinone/g of cells per second) - r _{O 2} consumption rate of O₂ (g oxygen/g of cells per second) - r _suc consumption rate of sucrose (g sucrose/g of cells per second) - V _m maximum respiration rate (g oxygen/g of cells per second) T. Nozawa is now with the Department of Agricultural Chemistry, University of TokyoT. Isohara is now with the Nippon Steel Corporation     

The rate of the deoxygenation reaction limits myoglobin- and hemoglobin-facilitated O₂ diffusion in cells

A mathematical model describing facilitation of O(2) diffusion by the diffusion of myoglobin and hemoglobin is presented. The equations are solved numerically by a finite-difference method for the conditions as they prevail in cardiac and skeletal muscle and in red cells without major simplifications. It is demonstrated that, in the range of intracellular diffusion distances, the degree of facilitation is limited by the rate of the chemical reaction between myglobin or hemoglobin and O(2). The results are presented in the form of relationships between the degree of facilitation and the length of the diffusion path on the basis of the known kinetics of the oxygenation-deoxygenation reactions. It is concluded that the limitation by reaction kinetics reduces the maximally possible facilitated oxygen diffusion in cardiomyoctes by ～50% and in skeletal muscle fibers by ～ 20%. For human red blood cells, a reduction of facilitated O(2) diffusion by 36% is obtained in agreement with previous reports. This indicates that, especially in cardiomyocytes and red cells, chemical equilibrium between myoglobin or hemoglobin and O(2) is far from being established, an assumption that previously has often been made. Although the "O(2) transport function" of myoglobin in cardiac muscle cells thus is severely limited by the chemical reaction kinetics, and to a lesser extent also in skeletal muscle, it is noteworthy that the speed of release of O(2) from MbO(2), the "storage function," is not limited by the reaction kinetics under physiological conditions.     

Differences in Hematological Traits between High- and Low-Altitude Lizards (Genus Phrynocephalus)

Phrynocephalus erythrurus (Lacertilia: Agamidae) is considered to be the highest living reptile in the world (about 4500-5000 m above sea level), whereas Phrynocephalus przewalskii inhabits low altitudes (about 1000-1500 m above sea level). Here, we report the differences in hematological traits between these two different Phrynocephalus species. Compared with P. przewalskii, the results indicated that P. erythrurus own higher oxygen carrying capacity by increasing red blood cell count (RBC), hemoglobin concentration ([Hb]) and hematocrit (Hct) and these elevations could promote oxygen carrying capacity without disadvantage of high viscosity. The lower partial pressure of oxygen in arterial blood (PaO₂) of P. erythrurus did not cause the secondary alkalosis, which may be attributed to an efficient pulmonary system for oxygen (O₂) loading. The elevated blood-O₂ affinity in P. erythrurus may be achieved by increasing intrinsic O₂ affinity of isoHbs and balancing the independent effects of potential heterotropic ligands. We detected one α-globin gene and three β-globin genes with 1 and 33 amino acid substitutions between these two species, respectively. Molecular dynamics simulation results showed that amino acids substitutions in β-globin chains could lead to the elimination of hydrogen bonds in T-state Hb models of P. erythrurus. Based on the present data, we suggest that P. erythrurus have evolved an efficient oxygen transport system under the unremitting hypobaric hypoxia.     

Effects of acclimation to altitude on oxygen affinity and organic phosphate concentrations in pigeon blood.

Hematocrit ratio, hemoglobin concentration and blood oxygen affinity, Bohr effect factor and Hill coefficient, adenosine triphosphate and inositol pentaphosphate (IPP) concentrations were studied in blood of adult pigeons exposed first at 140 m, and then for 3 weeks at 4000 m in an altitude chamber. At altitude, the hematocrit ratio and hemoglobin concentration significantly increased, IPP concentration decreased, and P₅₀ did not change. A lower mean red cell age and a higher hemoglobin concentration may account for the unchanged P₅₀. Adaptation to hypoxia of the tissue oxygen supply was shown by a greater blood O₂ capacitance (ΔC_HbO₂/Δ_o2) in the physiological range of oxygen partial pressures.     

The steady-state transport of oxygen through hemoglobin solutions

The steady-state transport of oxygen through hemoglobin solutions was studied to identify the mechanism of the diffusion augmentation observed at low oxygen tensions. A novel technique employing a platinum-silver oxygen electrode was developed to measure the effective diffusion coefficient of oxygen in steady-state transport. The measurements were made over a wider range of hemoglobin and oxygen concentrations than previously reported. Values of the Brownian motion diffusion coefficient of oxygen in hemoglobin solution were obtained as well as measurements of facilitated transport at low oxygen tensions. Transport rates up to ten times greater than ordinary diffusion rates were found. Predictions of oxygen flux were made assuming that the oxyhemoglobin transport coefficient was equal to the Brownian motion diffusivity which was measured in a separate set of experiments. The close correlation between prediction and experiment indicates that the diffusion of oxyhemoglobin is the mechanism by which steady-state oxygen transport is facilitated.     

Low oxygen affinity derivatives of human hemoglobin by fixation of polycarboxylic dextran to the oxyform

Solutions of modified adult human hemoglobin (Hb) have potential applications as physiological oxygen carriers. The chemical modification that has been the most studied during the last few years is the cross-linking of the protein between its two αβ dimers, in order, first, to hamper their diffusion through the kidney and therefore increase the plasma persistence of Hb, and second, to decrease its oxygen affinity. However, despite the cross-linking, the vascular retention time is only increased by a factor of three, and a supplementary modification of cross-linked Hb is needed in order to further improve its in vivo half-life. The Hb derivatives described in this paper were obtained by the covalent fixation of benzene tetracarboxylate-substituted dextran onto oxyHb. The resulting conjugates all exhibited a higher P₅₀ than native Hb. The experiments carried out in the presence of inositolhexaphosphate showed that the allosteric sites of Hb molecules were occupied by the polymeric reagent. The important decrease in the Bohr effect and the lack of the Cl^? effect on the oxygen-binding properties proved that the Val 1α residue was also substituted. Finally, the ability of some conjugates to unload as much O₂ as blood, together with their other properties, make them quite promising candidates as red cell substitutes. © 1993 John Wiley & Sons, Inc.     

Transient effects on the initial rate of oxygenation of red blood cells

The rate-controlling process in the oxygenation of red blood cells is investigated using a Roughton-like model for oxygen diffusion and reaction with hemoglobin. The mathematical equations describing the model are solved using two independent techniques, numerical inversions of the Laplace transform of the equations and numerical solutions via an implicit-explicit finite difference form of the equations. The model is used to re-examine previous theoretical models that incorporate either a red cell membrane that is resistive to oxygen diffusion or an unstirred layer of water surrounding the cell. Although both models have been postulated to be equivalent, the results of the computer simulations demonstrate significant differences between the two models in the rate of oxygenation of the red cells, depending upon the values chosen for the diffusion coefficient for O₂ in the membrane and the thickness of the water layer. The difference is apparently due to differences in the induction and transient periods of the water layer model relative to the membrane model.     

Anion transport in embryonic and adult chicken red blood cells

The rates of Cl and SO₄ transport at 0° and 37°C, respectively, have been measured under exchange conditions for red blood cells of embryonic and adult chickens. It was found that the rate of self-exchange of SO₄ in embryonic red cells decreases as the embryo matures, and that the SO₄ transport rate was lower in adult compared to embryonic red cells. In contrast, no difference in the rate of Cl self-exchange was found between adult and embryonic red cells.