Concentrated Hemoglobin Solution Research Articles

Prolonged functional life span of artificial red cells in blood circulation by repeated methylene blue injections

Hemoglobin-vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated hemoglobin solution in phospholipid vesicles (liposomes) and their safety and efficacy as a transfusion alternative have been evaluated. Because of the absence of enzymatic methemoglobin reduction system in HbV, the level of ferric methemoglobin (metHb) increases gradually after intravenous administration. Our previous studies clarified that the glycolytic electron energies, charged as NAD(P)H in red blood cells (RBC), are donated to reduce metHb compartmentalized in HbV via a water-soluble electron mediator such as methylene blue [MB; 3,7-bis(dimethylamino)phenothiazinium chloride], which freely shuttle across both RBC biomembrane and HbV lipid membrane. Herein, we tried to test repeated injections of MB after the massive HbV administration (28 mL/kg) to hemorrhagic shocked Wistar rats (n = 3). MB was injected (3.1 mg/kg) at 7, 24 and 48 h after HbV administration. Every MB injection showed rapid reduction of metHb and gradual reversal increase. As a result, the functional life span of HbV was significantly extended over 60 h. It is expected that further optimization of injection scheduling will decrease the total amount of MB and prolong the functional life span of HbV.

Transmembrane Difference in Colloid Osmotic Pressure Affects the Lipid Membrane Fluidity of Liposomes Encapsulating a Concentrated Protein Solution.

A hemoglobin vesicle (Hb-V) is an artificial oxygen carrier encapsulating a highly concentrated hemoglobin solution (40 g/dL) in a liposome. The in vivo safety and efficacy of Hb-V suspension as a transfusion alternative and structural stability during storage have been studied extensively. Because the intraliposomal Hb aqueous solution can possess colloid osmotic pressure (COP, 200-300 Torr) that is much higher than that of blood plasma (20-25 Torr), a question arises as to whether the lipid membrane senses the transmembrane difference in COP. We examined the membrane microviscosity using a fluorescence polarization technique. To avoid the interference of red Hb on the fluorescence measurement, we used human serum albumin (HSA) as a substitute for Hb. Both HSA and Hb solutions show high COP depending on the concentration. Encapsulation of HSA solution (40 g/dL) in the liposome decreased the membrane microviscosity at a lower temperature (949 ± 8 cP → 607 ± 10 cP at 25 °C). The result indicates that the transmembrane osmotic stress induced by HSA encapsulation expands the liposome maximally with increasing spherical surface area, and the membrane fluidity is increased extremely. Even for such a condition, the lowest membrane microviscosity, 377 ± 10 cP at 60 °C, is much higher than that of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine liposome (40 ± 2 cP at 60 °C). Accordingly, Hb-V as well as HSA-V maintains a spherical structure and mechanical stability under transmembrane stress caused by high COP, as described in the literature.

A Fourteen-Day Observation and Pharmacokinetic Evaluation after a Massive Intravenous Infusion of Hemoglobin-Vesicles (Artificial Oxygen Carriers) in Cynomolgus Monkeys

Hemoglobin-vesicles (HbV) are a cellular type hemoglobin-based oxygen carrier in which a concentrated hemoglobin solution is encapsulated within a phospholipid vesicle (liposome). Although it was previously revealed that HbV possesses a higher biocompatibility, low toxicity and no accumulation in the body in an animal model, these assessments were limited to the use of rodents, including mice, rats and rabbits as models. The aim of this study was to observe the effects of the administration of HbV in a nonhuman primate. For this purpose, cynomolgus monkeys were used as the model and the systematic response, serum biochemical analysis and pharmacokinetic properties were monitored for 14 days after a massive intravenous injection of HbV at a putative dose (1400 mg Hb/ kg, 17.5 mL/kg). All of the monkeys tolerated the massive amount of injected HbV and survived, and no abnormal behavior was observed. The systematic response and serum biochemical analysis were overall normal, except for a transient elevation in alanine aminotransferase levels. In addition, the levels of phospholipids, total cholesterol and total bilirubin, metabolites of hemoglobin and lipid components of HbV, were increased after HbV administration. In the pharmacokinetic study, HbV was retained for a sufficient period to permit it to function as an alternative to red blood cells and showed good metabolic properties without accumulation in the bloodstream. In conclusion, this is the first report of biological reactions and a pharmacokinetic evaluation for a 14 day period after a massive intravenous injection of HbV in a primate. The results obtained in this study provide useful information, not only for the development of further optimized HbV but also for designing relevant and rational protocols for clinical trials.

Removal of Cellular‐Type Hemoglobin‐Based Oxygen Carrier (Hemoglobin‐Vesicles) From Blood Using Centrifugation and Ultrafiltration

The hemoglobin-vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a phospholipid vesicle (liposome). During or after transporting oxygen, macrophages capture HbVs in the reticuloendothelial system (RES) with an approximate circulation half-life of 3 days. Animal studies show transient splenohepatomegaly after large doses, but HbVs were completely degraded, and the components were excreted in a few weeks. If a blood substitute is used for emergency use until red blood cell transfusion becomes available or for temporary use such as a priming fluid for an extracorporeal circuit, then one option would be to remove HbVs from the circulating blood without waiting a few weeks for removal by the RES. Using a mixture of beagle dog whole blood and HbV, we tested the separation of HbV using a centrifugal Fresenius cell separator and an ultrafiltration system. The cell separator system separated the layers of blood cell components from the HbV-containing plasma layer by centrifugal force, and then the HbV was removed from plasma phase by the ultrafiltration system. The HbVs (250-280 nm) are larger than plasma proteins (< 22 nm diameter) but smaller than blood cell components (> 3 µm). The size of HbVs is advantageous to be separated from the original blood components, and the separated blood components can be returned to circulation.

Repeated Injection of High Doses of Hemoglobin-Encapsulated Liposomes (Hemoglobin Vesicles) Induces Accelerated Blood Clearance in a Hemorrhagic Shock Rat Model

The hemoglobin vesicle (HbV) is an artificial oxygen carrier in which a concentrated hemoglobin solution is encapsulated in a liposome. To apply liposome preparations in clinics, it is important to consider the accelerated blood clearance phenomenon (ABC phenomenon), which involves a loss in the long-circulation half-life after being administered repeatedly to the same animals. The objective of this study was to determine whether the ABC phenomenon is induced by repeated injection of HbV under conditions of hemorrhagic shock. We created a rat model of hemorrhagic shock and performed a pharmacokinetic study using (125)I-HbV, in which the Hb inside of HbV was labeled with (125)I. At 4 and 7 days after resuscitation from hemorrhagic shock by nonlabeled HbV (1400 mg Hb/kg), the second dose of (125)I-HbV (1400 mg Hb/kg) was rapidly cleared from the circulation compared with normal rats. Of interest, IgM against HbV was produced at 4 days after the first injection of HbV, but decreased at 7 days. In addition, phagocyte activity was increased at both 4 and 7 days after the first injection of HbV. These results suggest that repeated injections of HbV at a dose of 1400 mg Hb/kg induce the ABC phenomenon under conditions of hemorrhagic shock, which is strongly related to both the production of anti-HbV IgM and enhanced phagocyte activity. We thus conclude that it might be necessary to consider the ABC phenomenon in the dose regimen of HbV treatment in clinical settings.

Observation of a Large-Scale Superstructure in Concentrated Hemoglobin Solutions by Using Small Angle Neutron Scattering

We studied the properties of highly concentrated hemoglobin solutions from 137 to 290 mg/mL using small angle neutron scattering over a very large momentum transfer range from q = 3.4 × 10−4 to 0.16 Å−1. The 137 and 227 mg/mL solutions revealed the presence of a large-scale superstructure with a radius of gyration on the order of 3000 Å. The 290 mg/mL solution was characterized by a mass fractal dimension of 2.67. Additionally, the scattering curve of the 290 mg/mL solution exhibited two shoulders, which might be due to the shape of the superstructure. In contrast, the radius of gyration of a single hemoglobin molecule is ∼24 Å.

Hemoglobin Vesicles, Polyethylene Glycol (PEG)ylated Liposomes Developed as a Red Blood Cell Substitute, Do Not Induce the Accelerated Blood Clearance Phenomenon in Mice

The hemoglobin vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a liposome of which the surface is covered with polyethylene glycol (PEG). It was recently reported that repeated injections of PEGylated liposomes induce the accelerated blood clearance (ABC) phenomenon, in which serum anti-PEG IgM plays an essential role. To examine this issue, we investigated whether HbV induces the ABC phenomenon in mice at a dose of 0.1 mg Hb/kg, a dose that is generally known to induce the ABC phenomenon, or at 1400 mg Hb/kg, which is proposed for clinical use. At 7 days after the first injection of nonlabeled HbV (0.1 mg Hb/kg), the mice received HbV in which the Hb had been labeled with (125)I. After a second injection, HbV was rapidly cleared from the circulation, and uptake clearances in liver and spleen were significantly increased. In contrast, at a dose of 1400 mg Hb/kg, the pharmacokinetics of HbV was negligibly affected by repeated injection. It is interesting to note that IgM against HbV was produced 7 days postinjection at both of the above doses, and their recognition site was determined to be 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG in HbV. These results suggest that a clinical dose of HbV does not induce the ABC phenomenon, and that suppression of ABC phenomenon is caused by the saturation of phagocytic processing by the mononuclear phagocyte system. Thus, we conclude that induction of the ABC phenomenon would not be an issue in the dose regimen used in clinical settings.

HEMOGLOBIN VESICLES AND RED BLOOD CELLS AS CARRIERS OF CARBON MONOXIDE PRIOR TO OXYGEN FOR RESUSCITATION AFTER HEMORRHAGIC SHOCK IN A RAT MODEL

Hemoglobin vesicles (HbVs) are artificial oxygen (O2) carriers that encapsulate concentrated hemoglobin (Hb) solution in phospholipid vesicles (liposomes). Recent reports on cytoprotective effects of exogenous carbon monoxide (CO) urged us to test infusion of CO-bound HbV (CO-HbV) and red blood cells (CO-RBC) in hemorrhagic-shocked rats to improve tissue viability over that of O2-bound HbV (O2-HbV) and O2-bound RBC (O2-RBC). Male Wistar rats were anesthetized with 1.5% sevoflurane inhalation (FiO2 = 21%) while spontaneous breathing was maintained. Shock was induced by 50% blood withdrawal from femoral artery. Fifteen minutes later, they received CO-HbV, CO-RBC, O2-HbV, O2-RBC, or empty vesicles (EV) suspended in 5% recombinant albumin. All groups showed prompt recovery of blood pressure and blood gas parameters just after resuscitation and survived for 6 h of observation period. However, only the EV group showed significant hypotension at 3 and 6 h. Plasma enzyme levels were elevated at 6 h, especially in the O2-HbV, O2-RBC, and EV groups. They were significantly lower in the CO-HbV and CO-RBC groups than in the O2-bound fluids. Immunohistochemical staining of 3-nitrotyrosine exhibited less oxidative damage in the liver and lung for CO-HbV and CO-RBC groups. Blood carbonyl Hb levels (26%-39% immediately after infusion) decreased to less than 3% at 6 h while CO was exhaled through the lung. Both HbV and RBC gradually gained the O2 transport function. Collectively, both CO-HbV and CO-RBC showed a resuscitative effect for hemorrhagic-shocked rats. They reduced oxidative damage to organs in comparison to O2-HbV and O2-RBC. Adverse and poisonous effects of CO gas were not evident for 6 h in this experimental model. Further study is necessary to clarify the neurological impact of a longer observation period for eventual clinical applications.

Encapsulation of Concentrated Hemoglobin Solution in Phospholipid Vesicles Retards the Reaction with NO, but Not CO, by Intracellular Diffusion Barrier

One physiological significance of the red blood cell (RBC) structure is that NO binding of Hb is retarded by encapsulation with the cell membrane. To clarify the mechanism, we analyzed Hb-vesicles (HbVs) with different intracellular Hb concentrations, [Hb](in), and different particle sizes using stopped-flow spectrophotometry. The apparent NO binding rate constant, k(on)('(NO)), of HbV at [Hb](in) = 1 g/dl was 2.6 x 10(7) m(-1) s(-1), which was almost equal to k(on)((NO)) of molecular Hb, indicating that the lipid membrane presents no obstacle for NO binding. With increasing [Hb](in) to 35 g/dl, k(on)('(NO)) decreased to 0.9 x 10(7) m(-1) s(-1), which was further decreased to 0.5 x 10(7) m(-1) s(-1) with enlarging particle diameter from 265 to 452 nm. For CO binding, which is intrinsically much slower than NO binding, k(on)('(CO)) did not change greatly with [Hb](in) and the particle diameter. Results obtained using diffusion simulations coupled with elementary binding reactions concur with these tendencies and clarify that NO is trapped rapidly by Hb from the interior surface region to the core of HbV at a high [Hb](in), retarding NO diffusion toward the core of HbV. In contrast, slow CO binding allows time for further CO-diffusion to the core. Simulations extrapolated to larger particles (8 mum) showing retardation even for CO binding. The obtained k(on)('(NO)) and k(on)('(NO)) yield values similar to those reported for RBCs. In summary, the intracellular, not extracellular, diffusion barrier is predominant due to the rapid NO binding that induces a rapid sink of NO from the interior surface to the core, retarding further NO diffusion and binding.

Sorption purification of concentrated hemoglobin solutions

A procedure was developed for purification of concentrated hemoglobin solutions from hemolyzate of human erythrocytes to remove soluble cellular impurities using a frontal chromatographic process on an anion exchanger under conditions of selective absorption of impurities and the absence of sorption of the target product, hemoglobin.

Structural transition temperature of hemoglobins correlates with species’ body temperature

Human red blood cells (RBCs) exhibit sudden changes in their biophysical properties at body temperature (T (B)). RBCs were seen to undergo a spontaneous transition from blockage to passage at T (C) = 36.4 +/- 0.3 degrees C, when the temperature dependency of RBC-passages through 1.3 mum narrow micropipettes was observed. Moreover, concentrated hemoglobin solutions (45 g/dl) showed a viscosity breakdown between 36 and 37 degrees C. With human hemoglobin, a structural transition was observed at T (B) as circular dichroism (CD) experiments revealed. This leads to the assumption that a species' body temperature occupies a unique position on the temperature scale and may even be imprinted in the structure of certain proteins. In this study, it was investigated whether hemoglobins of species with a T (B) different from those of human show temperature transitions and whether those were also linked to the species' T (B). The main conclusion was drawn from dynamic light scattering (DLS) and CD experiments. It was observed that such structural temperature transitions did occur in hemoglobins from all studied species and were correlated linearly (slope 0.81, r = 0.95) with the species' body temperature. We presumed that alpha-helices of hemoglobin were able to unfold more readily around T (B). alpha-helical unfolding would initiate molecular aggregation causing RBC passage and viscosity breakdown as mentioned above. Thus, structural molecular changes of hemoglobin could determine biophysical effects visible on a macroscopic scale. It is hypothesized that the species' body temperature was imprinted into the structure of hemoglobins.

Use of Hemoglobin Vesicles During Cardiopulmonary Bypass Priming Prevents Neurocognitive Decline in Rats

Homologous blood use is considered to be the gold standard for cardiopulmonary bypass (CPB) priming in infants despite exposure of the patient to potential cellular and humoral antigens. However, the use of hemoglobin vesicles (HbVs), artificial oxygen carriers that encapsulate a concentrated hemoglobin solution within phospholipid bilayer membranes, for CPB priming may prevent neurocognitive decline in infants. The goal of this study was to determine whether HbV use offsets hemodilution caused by patient/priming volume-mismatched CPB and thereby prevents the development of postoperative neurocognitive deficits. CPB was established in 28 male Sprague-Dawley rats (age, 14 to 16 weeks; weight, 450 grams) after cannulation of the tail artery and right atrium. The animals were randomly assigned to 1 of 3 groups: sham surgery (n=9), HbV (-) prime (n=10), or HbV (+) prime (n=9). CPB was conducted for 90 minutes at 200 mL/kg per minute. The hematocrit during CPB was 10.0+/-1.2% in the HbV (+) prime group and 9.9+/-1.3% in the HbV (-) prime group (P=not significant). Learning and memory function were evaluated using 2 different maze tests (Maze-1 and Maze-2, in which the arrival times to the target were measured on the first, third, fifth, and seventh postoperative days). Learning and memory function were significantly better in the HbV (+) prime group than in the HbV (-) prime group (Maze-1, P=0.012; Maze-2, P=0.042); there was no difference between the HbV (+) prime and the sham surgery group. The use of HbV for CPB priming may serve as a substitute for homologous blood to prevent the unacceptable hemodilution and contribute to maintenance of intact neurocognitive function.

Effects of Hemoglobin Vesicles on Resting and Agonist-Stimulated Human Platelets In Vitro

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alphaIIbbeta3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.

Determination of the complex refractive index of highly concentrated hemoglobin solutions using transmittance and reflectance measurements

The complex refractive index of highly concentrated hemoglobin solutions as they appear in red blood cells are determined in the wavelength range of 250 to 1100 nm using transmittance and Fresnel reflectance measurements. The determined real parts of the refractive indices are on average 0.02 units higher than the values found in the literature. The wavelength dependence of the measured data in the UV region differs from the calculated data using the Kramers-Kronig relation.

Reduction of methemoglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles.

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH(2) by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O(2) binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme (Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O(2) binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 microm (close to capillary diameter in tissue, 5 microm), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH(2) reacted with O(2) to produce H(2)O(2), which was detected by the fluorescence measurement of the reaction of H(2)O(2) and p-nitrophenylacetic acid. However, the amount of H(2)O(2) generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH(2) was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.

Allowance for thermodynamic non-ideality in the characterization of protein self-association by frontal exclusion chromatography: hemoglobin revisited

This investigation re-examines theoretical aspects of the allowance for effects of thermodynamic non-ideality on the characterization of protein self-association by frontal exclusion chromatography, and thereby provides methods of analysis with greater thermodynamic rigor than those used previously. Their application is illustrated by reappraisal of published exclusion chromatography data for hemoglobin on the controlled-pore-glass matrix CPG-120. The equilibrium constant of 100/M that is obtained for dimerization of the α 2β 2 species by this means is also deduced from re-examination of published studies of concentrated hemoglobin solutions by osmotic pressure and sedimentation equilibrium methods.

Development of a compact artificial gill using concentrated hemoglobin solution as the oxygen carrier

An artificial gill was developed using a concentrated hemoglobin solution containing inositol hexaphosphate (IHP), as the oxygen carrier solution, with the oxygen affinity controlled by temperature. Oxygen dissolved in sea water is first taken up from water to the oxygen carrier solution at 293 K. The oxygen carrier solution is then heated and the oxygen is released from the oxygen carrier solution to expired air at 310 K. The enhancement factors of the oxygen carrier solution that indicate its performance were obtained from the oxygen uptake rate and oxygen release rate. The values were approximately 3 at oxygen uptake and 16 at oxygen release. The scale-up for a human being at rest was estimated using these values, and the optimal operating condition was determined. The required membrane surface area for a human being is 63.8 m 2. The oxygen partial pressure of inspiration is 20.7 kPa, adequate for the respiration. These indicate the feasibility of a compact and portable artificial gill device.

Pretreatment of serum containing hemoglobin vesicles (oxygen carriers) to prevent their interference in laboratory tests.

Hemoglobin vesicles (HbV, diameter: 251 +/- 81 nm) are artificial oxygen (O2) carriers encapsulating concentrated hemoglobin (Hb) solution with phospholipid bilayer membrane, and their O2 transporting ability in vivo has been extensively studied. It is important to clarify the interference of the HbV suspension in clinical laboratory tests performed on serum and to establish a pretreatment method to avoid such an interference. The HbV suspension, acellular Hb solution ([Hb] = 10 g/dl) or saline, was mixed with a pooled human serum at various ratios up to 50 vol% ([Hb] = 5 g/dl), and the magnitude of the interference effect of HbV and Hb on 30 analytes was studied. The mixture of the HbV suspension and serum was ultracentrifuged (50,000 g, 20 min) to remove the HbV particles as precipitate, and the supernatant was analyzed and compared with the saline control group. The HbV particles were also removed by centrifugation (2,700 g, 30 min) in the presence of dextran (Mw 200 kDa). The HbV suspension showed considerable interference effects in most analytes. The majority of these effects was more serious than those of the acellular Hb solution. These findings are thought to be due to the light absorption of Hb in HbV and/or the light scattering generated in the suspension that interferes with the colorimetric and turbidimetric measurements. The components of HbV may also interfere with the chemical reactions of the studied assays. However, removal of the HbV from the supernatant diminished the interference in most of the assays: this is an advantage of HbV in comparison with acellular chemically modified Hb solutions.

Optimum control of oxygen affinity of hemoglobin as an oxygen carrier solution for an artificial gill

An artificial gill was developed using a concentrated hemoglobin solution as an oxygen carrier solution, with the oxygen affinity controlled by temperature. Oxygen affinity of the concentrated hemoglobin solution was optimized for the artificial gill by adding inositol hexaphosphate as an allosteric effector and varying the pH and temperature. The oxyhemoglobin dissociation curve shifted to the right with increased ratios of inositol hexaphosphate to hemoglobin, decreased pH values, and higher temperatures. The optimum ratio of inositol hexaphosphate to hemoglobin was 5 : 1. Because lower pH promotes proton oxidation of hemoglobin, the optimum pH was 6.9. The oxyhemoglobin dissociation curve shifted to the right with higher temperatures. The amount of the shift in relation to temperature change was higher at higher temperatures. The oxygen transfer rate was markedly increased by changing the temperature of the oxygen carrier solution. The overall mass transfer coefficient decreased at higher oxygen partial pressures due to the gentle oxyhemoglobin dissociation curve. The flow rate of the oxygen carrier solution and heat transfer rate for heating of the oxygen carrier solution were markedly decreased by setting the range of temperature changes from 293 K to 310 K. The flow rate of the oxygen carrier solution required to supply 300 ml (STP)/min of oxygen was 21.7 l/min, and the heat transfer rate was 1165 kJ/min.

Temperature Transition of Human Hemoglobin at Body Temperature: Effects of Calcium

We studied the effects of calcium ion concentration on the temperature dependence of rheological behavior of human red blood cells (RBCs) and concentrated hemoglobin solutions. Our previous study (G. M. Artmann, C. Kelemen, D. Porst, G. Büldt, and S. Chien, 1998, Biophys. J., 75:3179–3183) showed a critical temperature ( T c) of 36.4 ± 0.3°C at which the RBCs underwent a transition from non-passage to passage through 1.3- μm micropipettes in response to an aspiration pressure of −2.3 kPa. An increase in intracellular Ca 2+ concentration by using the ionophore A23187 reduced the passability of intact RBCs through small micropipettes above T c; the micropipette diameter needed for >90% passage increased to 1.7 μm. Viscometry of concentrated hemoglobin solutions (45 and 50 g/dl) showed a sudden viscosity transition at 36 ± 1°C ( T c η ) at all calcium concentrations investigated. Below T c η , the viscosity value of the concentrated hemoglobin solution at 1.8 mM Ca 2+ was higher than that at other concentrations (0.2 μM, 9 mM, and 18 mM). Above T c η , the viscosity was almost Ca 2+ independent. At 1.8 mM Ca 2+ and 36 ± 1°C, the activation energy calculated from the viscometry data showed a strong dependence on the hemoglobin concentration. We propose that the transition of rheological behavior is attributable to a high-to-low viscosity transition mediated by a partial release of the hemoglobin-bound water.