Extracellular hemoglobin combined with an O2-generating material overcomes O2 limitation in the bioartificial pancreas

The bioartificial pancreas encapsulating pancreatic islets in immunoprotective hydrogel is a promising therapy for Type 1 diabetes. As pancreatic islets are highly metabolically active and exquisitely sensitive to hypoxia, maintaining O₂ supply after transplantation remains a major challenge. In this study, we address the O₂ limitation by combining silicone-encapsulated CaO₂ (silicone-CaO₂) to generate O₂ with an extracellular hemoglobin O₂-carrier coencapsulated with islets. We showed that the hemoglobin improved by 37% the O₂-diffusivity through an alginate hydrogel and displayed antioxidant properties neutralizing deleterious reactive O₂ species produced by silicone-CaO₂. While the hemoglobin alone failed to maintain alginate macroencapsulated neonate pig islets under hypoxia, silicone-CaO₂ alone or combined to the hemoglobin restored islet viability and insulin secretion and prevented proinflammatory metabolism (PTGS2 expression). Interestingly, the combination took the advantages of the two individual strategies, improved neonate pig islet viability and insulin secretion in normoxia, and VEGF secretion and PDK1 normalization in hypoxia. Moreover, we confirmed the specific benefits of the combination compared to silicone-CaO₂ alone on murine pseudo-islet viability in normoxia and hypoxia. For the first time, our results show the interest of combining an O₂ provider with hemoglobin as an effective strategy to overcome O₂ limitations in tissue engineering.     

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H₂O₂ using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H₂O₂ produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H₂O₂. © 1997 John Wiley & Sons, Ltd.     

Mathematical studies of the interaction of respiratory gases with whole blood II. CO2 absorption

Some progress has been made on the problem of the interaction of respiratory gases with whole blood. A working mathematical model for the O₂−CO₂ interaction phenomena has been developed from mathematical studies of the data. The Edsall-Wyman (1958) model for CO₂ absorption is improved upon in this paper by consolidating it with the O₂ absorption model developed in paper I of this set (Bernard, S. R.,Bull. Math. Biophysics,22, 391–415, 1960). This improved model assumed the effect of O₂ on CO₂ absorption is mediated through the electrical charge possessed by the hemoglobin molecule,i.e., O₂ molecules bound to hemoglobin displace protons from the hemoglobin thereby increasing the negative charge on the hemoglobin and at the same time increasing the acidity of the solution. The model is tested against the data.     

Transport of gases through hemoglobin solution

A mathematical theory is developed to explain the observed enhancement of O₂ transport through solutions by hemoglobin. At high partial pressures of O₂, ordinary diffusion through the solvent accounts for all transport of O₂, but at low partial pressures the transport may be increased manyfold by the presence of hemoglobin. This phenomenon is explained and its possible role in living organisms is discussed. The theory also indicates a new method of determining dissociation curves from diffusion experiments.     

Expression and Properties of Recombinant HbA2 (α2δ2) and Hybrids Containing δ-β Sequences

Hemoglobin A₂ (α₂δ₂), which is present at low concentration (1–2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O₂ binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A₂ and systematically studying its functional properties. The construct expressing HbA₂ contains only α and δ genes so that the extensive purification required to isolate natural HbA₂ is circumvented. Although natural hemoglobin A₂ is expressed at low levels in vivo, the amount of recombinant α₂δ₂ expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the α + β or the α + γ genes, respectively, are present on the construct. Recombinant HbA₂ is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA₂ are described.     

Dependence of oxygen uptake on ambient PO 2 in isolated perfused frog skin

Summary Rates of O₂ uptake across isolated perfused skin of bullfrogs (Rana catesbeiana) were measured in relation to blood flow at three levels of ambient O₂ tension: normoxia (O₂ tension=152 torr), hypoxia (12% O₂, 87 torr) and hyperoxia (42% O₂, 306 torr). At bulk perfusion rates ranging from 3.4 to 10.1 l·cm^-2·min^-1, O₂ uptake was positively correlated with hemoglobin delivery rate in both normoxia and hyperoxia, but was independent of delivery rate in hypoxia. Mean O₂ uptake in normoxia was 3.8 nmol O₂·cm^-2·min^-1 at a delivery rate of 9.8 nmol·cm^-2·min^-1 and 6.5 nmol O₂·cm^-2·min^-1 at a delivery rate of 28.3 nmol·cm^-2·min^-1. At any given bulk perfusion rate, oxygen uptake averaged about 49% lower in hypoxia than in normoxia, decreasing in proportion to the reduction of O₂ tension difference between medium and blood. In hyperoxia, O₂ uptake did not increase proportionally with the difference in O₂ tension between blood and medium, averaging only 50% higher at a 2.4-fold greater O₂ tension difference. Cutaneous diffusing capacity for O₂ averaged 0.041 nmol O₂·cm^-2·torr^-1·min^-1 during the first hour of perfusion in normoxia, and was not affected by reduction of ambient O₂ tension. The results indicate that cutaneous O₂ uptake in hypoxia is highly diffusion limited, and consequently, increases in cutaneous perfusion can not effectively compensate for reduction of ambient O₂ tension. In hyperoxia, O₂ uptake may be substantially perfusion limited because of reduced blood O₂ capacitance at high O₂ saturations.Abbreviations O₂ capacitance - C _Hb hemoglobin concentration - D diffusing capacity - PO₂ medium-blood PO₂ difference - Hb flow, hemoglobin delivery rate - Hepes N-[2-Hydroxyethyl]piperacine-N-[2 ethanesulfonic acid] - L _diff extent of diffusion limitation - MO₂ oxygen uptake rate - PO₂ oxygen tension - S O₂ saturation     

Steady-State, Hemoglobin-Facilitated O2 Transport in Human Erythrocytes

We measured the rate of oxygen transport through thin (165 µ) films of packed erythrocytes (Hb concentration = 30 g/100 ml). Under optimal conditions steady-state O₂ diffusion was nearly three times that found when the hemoglobin was prevented from acting as a carrier molecule by carbon monoxide binding or high oxygen back pressure. After each experiment we measured hemolysis and found that it averaged less than 1%. Hemolysis could not account for the facilitation, thus proving that facilitated transport of O₂ by hemoglobin can occur in red blood cells. The rate of facilitated transport was identical for Hb solutions of equal concentration to the cells. We interpret this to mean that under the conditions of our experiments the red cell membrane offers no detectable diffusion resistance to O₂ and that the mobility of Hb in intact red cells is the same as in concentrated Hb solution.     

The role of the superoxide anion as a toxic species in the erythrocyte.

The toxic action of the superoxide anion (O₂^?) toward the erythrocyte was investigated with O₂^? generated through the autooxidation of dihydroxyfumaric acid (DHF). A suspension of human red cells exposed to DHF undergoes a rapid breakdown of the cellular hemoglobin to methemoglobin and other green pigments. This hemoglobin breakdown is inhibited by superoxide dismutase (SOD) or catalase (CAT) and is accelerated by lactoperoxidase (LP) added externally to the red cell medium. Associated with the hemoglobin breakdown is a hypotonic hemolysis also inhibited by SOD or CAT and initially accelerated but later inhibited by LP. Conversion of the red cell hemoglobin to carbonmonoxyhemoglobin in an aerated medium results in no hemoglobin breakdown or hypotonic lysis in the presence of DHF, even though O₂^? can be demonstrated in the medium. Although no evidence for membrane sulfhydryl oxidation or lipid peroxidation can be demonstrated in red cells exposed to DHF, the membranes of these cells were found to retain a green pigment. The presence of this green pigment in red cell membranes was inhibited by SOD, CAT, or conversion of the cellular hemoglobin to carbonmonoxyhemoglobin, but was not inhibited by LP. These results have been interpreted as a peroxide-dependent formation of O₂^? by DHF, followed by attack of O₂^? on hemoglobin. The reaction of O₂^? with hemoglobin leads to the formation of a hemoglobin-breakdown product that binds to the red cell membrane, resulting in an increased osmotic fragility of the cell.     

Molecular oxygen binding with α and β subunits within the R quaternary state of human hemoglobin in solutions and porous sol–gel matrices

Nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O₂) rebinding to α and β subunits within oxygenated human adult hemoglobin in solutions and porous wet sol–gel matrices. Plasticity associated with the tertiary structure within R-state hemoglobin is explored through measurements that focus on the functional properties of hemoglobin under conditions designed to tune the tertiary structure without inducing the R to T transition. Inequivalence in the O₂ binding to the α and β hemes within the R quaternary structure is studied. The individual kinetic properties of the α and β subunits within the hemoglobin encapsulated in sol–gels and aged as the oxy derivative are shown to be independent of proton concentration over the pH range from 6.3 to 8.5. However, buffer effects on the subunits' properties are revealed in sol–gel-free mediums. Interestingly, the α and β subunits within the encapsulated hemoglobin possess the O₂ rebinding properties which fall within the range of the ones for oxygenated hemoglobin in the buffer solutions. The combined results show a pattern in which there is a progression of functional properties that are ascribed to a family of conformational substates of R-state hemoglobin. O₂ rebinding to the α and β subunits within the oxygenated R-state hemoglobin in both solutions and wet sol–gels is revealed to be modulated by tertiary structural changes in two quite different ways. The possible structural changes, which modify the O₂ rebinding properties, are discussed.     

Oxidation of hemoglobin by arenediazonium salts. The influence of dioxygen

The reactions of human hemoglobin with p-nitro- and p-chlorobenzenediazonium tetrafluoroborates in the presence and absence of molecular oxygen have been investigated in kinetic detail. The oxidation of iron(II) occurs with first order rate dependence on both the hemoglobin and diazonium salt concentrations, but inverse first order dependence on the concentration of molecular oxygen characterizes reactions performed in the presence of O₂. In the absence of O₂, nitrobenzene is the only product observed from hemoglobin oxidation by p-NO₂C₆H₄N₂⁺BF₄^?, and a 1:1 stoichiometry exists between nitrobenzene produced and Fe(II) oxidized. In the presence of O₂, p-nitrophenol is the dominant product, but product yield is dependent on the ratio of reactants. Electron transfer to the diazonium salt rather than its corresponding diazohydroxide or diazoate is inferred from the relative absence of pH dependence on the rate of oxidation. The composite results are consistent with a mechanism for hemoglobin oxidation that requires molecular oxygen dissociation from oxyhemoglobin prior to oxidation by the diazonium salt. Implications of this investigation for the mechanism of arylhydrazine reactions with hemoglobin are discussed.     

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     

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H₂O₂, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H₂O₂ or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.     

Hemoglobin‐based oxygen carrier and convection enhanced oxygen transport in a hollow fiber bioreactor

A mathematical model was developed to study O₂ transport in a convection enhanced hepatic hollow fiber (HF) bioreactor, with hemoglobin‐based O₂ carriers (HBOCs) present in the flowing cell culture media stream of the HF lumen. In this study, four HBOCs were evaluated: PEG‐conjugated human hemoglobin (MP4), human hemoglobin (hHb), bovine hemoglobin (BvHb) and polymerized bovine hemoglobin (PolyBvHb). In addition, two types of convective flow in the HF extra capillary space (ECS) were considered in this study. Starling flow naturally occurs when both of the ECS ports are closed. If one of the ECS ports is open, forced convective flow through the ECS will occur due to the imposed pressure difference between the lumen and ECS. This type of flow is referred to as cross‐flow in this work, since some of the fluid entering the HF lumen will pass across the HF membrane and exit via the open ECS port. In this work, we can predict the dissolved O₂ concentration profile as well as the O₂ transport flux in an individual HF of the bioreactor by solving the coupled momentum and mass transport equations. Our results show that supplementation of the cell culture media with HBOCs can dramatically enhance O₂ transport to the ECS (containing hepatocytes) and lead to the formation of an in vivo‐like O₂ spectrum for the optimal culture of hepatocytes. However, both Starling flow and cross‐flow have a very limited effect on O₂ transport in the ECS. Taken together, this work represents a novel predictive tool that can be used to design or analyze HF bioreactors that expose cultured cells to defined overall concentrations and gradients of O₂. Biotechnol. Bioeng. 2009;102: 1603–1612. © 2008 Wiley Periodicals, Inc.     

Functional diversification of sea lamprey globins in evolution and development

Agnathans have a globin repertoire that markedly differs from that of jawed (gnathostome) vertebrates. The sea lamprey (Petromyzon marinus) harbors at least 18 hemoglobin, two myoglobin, two globin X, and one cytoglobin genes. However, agnathan hemoglobins and myoglobins are not orthologous to their cognates in jawed vertebrates. Thus, blood-based O₂ transport and muscle-based O₂ storage proteins emerged twice in vertebrates from a tissue-globin ancestor. Notably, the sea lamprey displays three switches in hemoglobin expression in its life cycle, analogous to hemoglobin switching in vertebrates. To study the functional changes associated with the evolution and ontogenesis of distinct globin types, we determined O₂ binding equilibria, type of quaternary assembly, and nitrite reductase enzymatic activities of one adult (aHb5a) and one embryonic/larval hemoglobin (aHb6), myoglobin (aMb1) and cytoglobin (Cygb) of the sea lamprey. We found clear functional differentiation among globin types expressed at different developmental stages and in different tissues. Cygb and aMb1 have high O₂ affinity and nitrite reductase activity, while the two hemoglobins display low O₂ affinity and nitrite reductase activity. Cygb and aHb6 but not aHb5a show cooperative O₂ binding, correlating with increased stability of dimers, as shown by gel filtration and molecular modeling. The high O₂-affinity and the lack of cooperativity confirm the identity of the sea lamprey aMb1 as O₂ storage protein of the muscle. The dimeric structure and O₂-binding properties of sea lamprey and mammalian Cygb were very similar, suggesting a conservation of function since their divergence around 500 million years ago.     

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.     

A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV–vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0–10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H₂O₂ without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H₂O₂ in the range 5 × 10⁻⁷–3 × 10⁻⁴ M with a detection limit of 6 × 10⁻⁸ M. The response shows Michaelis–Menten behavior at higher H₂O₂ concentrations and the apparent Michaelis–Menten constant is estimated to be 112 μM.     

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H₂O₂ in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H₂O₂ source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H₂O₂ is sensitive to intracellular H₂O₂ competitors: it decreases when H₂O₂ is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H₂O₂. Oxidation of the fluorescent probes following reaction of H₂O₂ with endogenous HRP overcomes most criticisms in their use for intracellular H₂O₂ detection. The method can be applied for direct determination in plate reader and is proposed to detect H₂O₂ generation in physio-pathological cell models.     

Expression and properties of recombinant HbA2 (alpha2delta2) and hybrids containing delta-beta sequences

Hemoglobin A₂ (₂₂), which is present at low concentration (1–2%) in the circulating red cells of normal individuals, has two important features that merit its study, i.e., it inhibits polymerization of sickle HbS and its elevated concentration in some thalassemias is a useful clinical diagnostic. However, reports on its functional properties regarding O₂ binding are conflicting. We have attempted to resolve these discrepancies by expressing, for the first time, recombinant hemoglobin A₂ and systematically studying its functional properties. The construct expressing HbA₂ contains only and genes so that the extensive purification required to isolate natural HbA₂ is circumvented. Although natural hemoglobin A₂ is expressed at low levels in vivo, the amount of recombinant ₂₂ expressed in yeast is similar to that found for adult hemoglobin A and for fetal hemoglobin F when the + or the + genes, respectively, are present on the construct. Recombinant HbA₂ is stable, i.e., not easily oxidized, and it is a cooperative functional hemoglobin with tetramer-dimer dissociation properties like those of adult HbA. However, its intrinsic oxygen affinity and response to the allosteric regulators chloride and 2,3-diphosphoglycerate are lower than the corresponding properties for adult hemoglobin. Molecular modeling studies which attempt to understand these properties of HbA₂ are described.     

Unusual peroxidase activity of polynitroxylated pegylated hemoglobin: Elimination of H2O2 coupled with intramolecular oxidation of nitroxides

Polynitroxylated hemoglobin (Hb(AcTPO)₁₂) has been developed as a hemoglobin-based oxygen carrier. While Hb(AcTPO)₁₂ has been shown to exert beneficial effects in a number of models of oxidative injury, its peroxidase activity has not been characterized thus far. In the blood stream, Hb(AcTPO)₁₂ undergoes reduction by ascorbate to its hydroxylamine form Hb(AcTPOH)₁₂. Here we report that Hb(AcTPOH)₁₂ exhibits peroxidase activity where H₂O₂ is utilized for intramolecular oxidation of its TPOH residues to TPO. This represents an unusual redox-catalytic mechanism whereby reduction of H₂O₂ is achieved at the expense of reducing equivalents of ascorbate converted into those of Hb(AcTPOH)₁₂, a new propensity that cannot be directly associated with ascorbate.     

The fluxes of H2O2 and O2 can be used to evaluate seed germination and vigor of Caragana korshinskii

Seed deterioration is detrimental to plant germplasm conservation, and predicting seed germination and vigor with reliability and sensitivity means is urgently needed for practical problems. We investigated the link between hydrogen peroxide (H₂O₂) flux, oxygen influx and seed vigor of Caragana korshinskii by the non-invasive micro-test technique (NMT). Some related physiological and biochemical changes in seeds were also determined to further explain the changes in the molecular fluxes. The results showed that there was a good linear relationship between germination and H₂O₂ flux, and that O₂ influx was more suitable for assessing seed vigor. H₂O₂ flux changed relatively little initially, mainly affected by antioxidants (APX, CAT and GSH) and H₂O₂ content; afterward, the efflux increased more and more rapidly due to high membrane permeability. With the damage of mitochondrial respiration and membrane integrity, O₂ influx was gradually reduced. We propose that monitoring H₂O₂ and O₂ fluxes by NMT may be a reliable and sensitive method to evaluate seed germination and vigor.