Numerical simulation of oxygen delivery to muscle tissue in the presence of hemoglobin-based oxygen carriers

This work represents a culmination of research on oxygen transport to muscle tissue, which takes into account oxygen transport due to convection, diffusion, and the kinetics of simultaneous reactions between oxygen and hemoglobin and myoglobin. The effect of adding hemoglobin-based oxygen carriers (HBOCs) to the plasma layer of blood in a single capillary surrounded by muscle tissue based on the geometry of the Krogh tissue cylinder is examined for a range of HBOC oxygen affinity, HBOC concentration, capillary inlet oxygen tension (pO(2)), and hematocrit. The full capillary length of the hamster retractor muscle was modeled under resting (V(max) = 1.57 x 10(-4) mLO(2) mL(-1) s(-1), cell velocity (v(c)) = 0.015 cm/s) and working (V(max) = 1.57 x 10(-3) mLO(2) mL(-1) s(-1), v(c) = 0.075 cm/s) conditions. Two spacings between the red blood cell (RBC) and the capillary wall were examined, corresponding to a capillary with and without an endothelial surface layer. Simulations led to the following conclusions, which lend physiological insight into oxygen transport to muscle tissue in the presence of HBOCs: (1) The reaction kinetics between oxygen and myoglobin in the tissue region, oxygen and HBOCs in the plasma, and oxygen and RBCs in the capillary lumen should not be neglected. (2) Simulation results yielded new insight into possible mechanisms of oxygen transport in the presence of HBOCs. (3) HBOCs may act as a source or sink for oxygen in the capillary and may compete with RBCs for oxygen. (4) HBOCs return oxygen delivery to muscle tissue to normal for varying degrees of hypoxia (inlet capillary pO(2) < 30 mmHg) and anemia (hematocrit < 46%) for the hamster model.     

Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms

Adding antioxidant activities to hemoglobin-based oxygen carriers (HBOCs) represents a means of reducing cell-free hemoglobin-mediated oxidative cascades. We have covalently bound nitroxides, a class of antioxidant enzyme mimetics, to HBOCs. The objectives of this study were (1) to evaluate the pharmacokinetic (PK) effects of administering nitroxide covalently bound to HBOCs compared to those of free nitroxide coadministered with HBOCs and (2) to elucidate the effects of differing molecular weight HBOCs on the PK of bound nitroxide in a conscious guinea pig model of 25% blood exchange transfusion. Two HBOC platforms were used, intramolecular cross-linked hemoglobin (XLHb) and dextran polymerized/conjugated XLHb (PolyHb). Polynitroxylation was achieved by reacting 4-(2-bromoacetamido)-2,2,6,6,-tetramethylpiperidine-1-oxyl with XLHb or PolyHb to form polynitroxylated XLHb and polynitroxylated PolyHb, respectively, whereas a physical mixture of XLHb or PolyHb with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl was prepared to reflect a molar equivalence to HBOC-bound nitroxide. Plasma concentrations of two redox states, nitroxide and hydroxylamine, were determined by electron paramagnetic resonance spectroscopy. Results are presented to illustrate the influence of covalent labeling and HBOC molecular weight on nitroxide PK. The therapeutic potential of polynitroxylation of HBOCs as it relates to observations from the current and previously reported studies is discussed.     

Biological response of human aortic endothelial cells exposed to acellular hemoglobin solutions developed as potential blood substitutes

The cardiovascular effects of hemoglobin-based oxygen carriers (HBOCs) are mainly related to their nitric oxide (NO) scavenging properties but other effects such as the impact of these hemoglobins on the endothelial cell (EC) biology are not well understood. We hypothesized that HBOCs could modify EC functions by altering gene expression, in particular the endothelial NO synthase (NOS3) and/or by activating EC. Cultured human aortic endothelial cells (HAEC) were incubated for 3 hours with purified cell-free Hb, Dex-BTC-Hb or alpha alpha-Hb (16 g/L). Expression of NOS3 mRNA and protein were assessed by semi-quantitative RT-PCR and Western blot respectively immediately after and 24 hours after incubation. The expression and localization of the adhesion molecule ICAM-1 were detected by fluorescence microscopy. None of the solutions tested modified NOS3 mRNA and protein expression despite adequate controls that up- or down-regulate NOS3 expression. The expression and the localization of ICAM-1 on the cell membrane were modified after 3 hours of incubation with all the hemoglobin solutions tested in a manner similar to tumor necrosis factor-alpha. In conclusion, HAEC incubation with clinically relevant concentrations of HBOCs induced changes in the pattern of ICAM-1 expression consistent with cell activation/cell signaling mechanisms. However, HBOCs did not alter NOS3 gene expression.     

Allosteric effects on oxidative and nitrosative reactions of cell-free hemoglobins

A review of the oxidative and nitrosative reactions of cell-free hemoglobin-based oxygen carriers (HBOCs) shows that these reactions are intimately linked and are subject to allosteric control. Cross-linking reactions used to produce HBOCs introduce conformational constraints and result in Hbs with reduced responses to heterotropic and homotropic allosteric effectors. The Nernst plots of heme oxidation of cross-linked HBOCs are shifted to higher potentials relative to unmodified Hb in the absence of allosteric effectors, in accord with their T-state stabilization and right-shifted Hill plots of O(2) binding. They exhibit enhanced rates of autoxidation and nitrite-induced oxidation, features that appear due to their having more solvent-accessible heme pockets. The stability of their NO-Hb derivatives varies as a result of allosteric effects on the extent of formation of pentacoordinate NO-heme geometry by alpha chains and subsequent oxidation of partner beta chains. The physiological implications of these findings on the safety, efficacy and design of second generation HBOCs are discussed in the framework of a reaction scheme showing linkages between Hb-mediated redox reactions. These redox reactions can drive formation of SNO-Hb and other reactive species and are of significance for the use of cell-free Hbs in vivo.     

Hemoglobin-based O2 carrier O2 affinity and capillary inlet pO2 are important factors that influence O2 transport in a capillary

Hemopure (Biopure; Cambridge, MA) and PolyHeme (Northfield Laboratories; Evanston, IL) are two acellular hemoglobin-based O2 carriers (HBOCs) currently in phase III clinical trials for use as red blood cell substitutes. The most common adverse side effect that these HBOCs exhibit is increased vasoconstriction. Autoregulatory theory has been presented as a possible explanation for this physiological effect, where it is hypothesized that low-affinity HBOCs over-deliver O2 to tissues surrounding arterioles, thereby eliciting vasoconstriction. In this paper, we wanted to investigate HBOC oxygenation of tissue surrounding a capillary, which is the smallest element of the circulatory system. An a priori model has been developed in which the performance of mixtures of acellular HBOCs (synthesized by our group and others) and human red blood cells (hRBCs) has been simulated using a Krogh tissue cylinder model (KTCM) comprising a capillary surrounded by a capillary membrane and skeletal muscle tissue in cylindrical coordinates with specified tissue O2 consumption rates and Michaelis-Menten kinetics. In this study, the total hemoglobin (hRBCs and HBOCs) concentration was kept constant. The HBOCs studied possessed O2 affinities that were higher and lower compared to hRBCs (P50's spanned 5-55 mmHg), and the equilibrium binding/release of oxygen to/from the HBOCs was modeled using the Adair equation. At normoxic inlet pO2's, there was no correlation between O2 flux out of the capillary and the O2 affinity of the HBOC. However, a correlation was found between the average pO2 tension in the capillary and the O2 affinity of the HBOC. Additionally, we studied the change in the O2 equilibrium curve of HBOCs with different O2 affinities over a wide range of inlet pO2's and found that changing the inlet pO2 greatly affected which HBOC, having a unique O2 affinity, best delivered O2 to the surrounding tissue. The analysis of oxygen transport presented could lead to a better prediction of which acellular HBOC is best suited for a specific transfusion application that many times depends on the capillary inlet pO2 tension.     

Consequences of chemical modifications on the free radical reactions of human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA0. As previously reported, exposure of oxyhemoglobin to H2O2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 microM HbA (18.0 +/- 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.     

High O2 affinity hemoglobin-based oxygen carriers synthesized via polymerization of hemoglobin with ring-opened 2-chloroethyl-beta-D-fructopyranoside and 1-o-octyl-beta-D-glucopyranoside

Second generation hemoglobin-based O(2) carriers (HBOCs) are being developed with high O(2) affinity (low P(50)) in order to suppress vasoconstriction elicited by over-oxygenating tissues, a problem associated with low O(2) affinity first generation HBOCs. Our group has previously investigated the polymerization of hemoglobin (Hb) with dialdehydes as a strategy for engineering high O(2) affinity HBOCs. In this study, two novel reactive dialdehydes were synthesized by ring-opening 2-chloroethyl-beta-D-fructopyranoside (2-CEFP) and 1-o-octyl-beta-D-glucopyranoside (1-OGP) at the 1,2-diol position, respectively, to yield novel Hb polymerizing reagents. High-affinity polymerized HBOCs were synthesized by reacting R-state bovine hemoglobin (bHb) with ring-opened 2-CEFP and 1-OGP at cross-linker to bHb molar ratios ranging from 10:1 to 30:1. The resulting polymerized bovine HBOCs (bHBOCs) displayed P(50)s ranging from 7 to 18 mmHg, cooperativities ranging from 0.8 to 1.4, and methemoglobin (metHb) levels ranging from 3% to 10%. The cross-linking reaction also stabilized the third stepwise Adair coefficient for bHbs reacted with ring-opened 1-OGP at cross-linker to bHb molar ratios of 20:1 and 30:1 and for bHbs reacted with ring-opened 2-CEFP at molar ratios of 30:1. Additionally, the number-averaged molecular weight, M(n), of each polymerized bHBOC was larger compared to bHb. Molecular weight distributions leaning towards larger molecular weight bHBOCs were obtained by increasing the cross-linker to bHb molar ratio. Taken together, the results of this study have identified novel Hb polymerization reagents that are easy to synthesize, and that are capable of yielding bHBOCs with higher O(2) affinities and weight-averaged molecular weights compared to bHb.     

Model of nitric oxide diffusion in an arteriole: impact of hemoglobin-based blood substitutes

Administration of hemoglobin-based oxygen carriers (HBOCs) frequently results in vasoconstriction that is primarily attributed to the scavenging of endothelium-derived nitric oxide (NO) by cell-free hemoglobin. The ensuing pressor response could be caused by the high NO reactivity of HBOC in the vascular lumen and/or the extravasation of hemoglobin molecules. There is a need for quantitative understanding of the NO interaction with HBOC in the blood vessels. We developed a detailed mathematical model of NO diffusion and reaction in the presence of an HBOC for an arteriolar-size vessel. The HBOC reactivity with NO and degree of extravasation was studied in the range of 2-58 x 10(6) M(-1) x s(-1) and 0-100%, respectively. The model predictions showed that the addition of HBOC reduced the smooth muscle (SM) NO concentration in the activation range (12-28 nM) for soluble guanylate cyclase, a major determinant of SM contraction. The SM NO concentration was significantly reduced when the extravasation of HBOC molecules was considered. The myoglobin present in the parenchymal cells scavenges NO, which reduces the SM NO concentration.     

All hemoglobin-based oxygen carriers are not created equally

Hemoglobin (Hb)-based oxygen carriers (HBOCs) also known as "blood substitutes" have been under active clinical development over the last two decades. Cell-free Hb outside its natural protective red blood cell environment, as is the case with all HBOCs, has been shown to be vasoactive in part due to the scavenging of vascular endothelial nitric oxide (NO) and may in some instances induce heme-mediated oxidative stress. Chemical modification intended to stabilize HBOCs in the tetrameric or polymeric forms introduces conformational constraints that result in proteins with diverse allosteric responses as well as oxidative and nitrosative redox side reactions. Intra and inter-molecular cross-linking may in some instances also determine the interactions between HBOCs and normal oxidative inactivation and clearance mechanisms. Oxygen and oxidative reactions of normal and several cross-linked Hbs as well as their interactions with endogenous plasma protein (haptoglobin) and cellular receptor pathways (macrophage CD163) differ significantly. Therefore, safety and efficacy may be addressed by designing HBOCs with modifications that limit hypertension, minimize heme destabilization and take into account endogenous Hb removal mechanisms to optimize exposure times for a given indication.     

Size‐dependent interaction of cells and hemoglobin–albumin based oxygen carriers prepared using the SPG membrane emulsification technique

Hemoglobin‐based oxygen carriers (HBOCs) of various sizes have been developed so far, but their optimum size has not been clarified yet. Here, we examined the effect of HBOCs size on their interaction with cells using Shirasu porous glass (SPG) membrane emulsification technique, which enables precise tuning of particle size. Microspheres composed of bovine hemoglobin (bHb) and bovine serum albumin (BSA) was fabricated with the average diameters of 1.2–18.3 μm and the coefficient of variation of below 13%. Cellular uptake of the microspheres by RAW264.7 was observed at a diameter below 5 μm; however, uptake of the microspheres by HepG2 and HUVEC were not observed at any diameter. No enhancement of the generation of reactive oxygen species in the cytoplasm was detected at diameters above 9.8 μm in the three cell lines, due to their low cellular uptake. In addition, cytotoxicity of the microspheres decreased with increasing microsphere diameter in the three cell lines and microspheres of 18.3 μm showed good cellular compatibility regardless of the oxyhemoglobin percentage. Since cytotoxicity is a crucial factor in their applications, our systemic investigation would provide a new insight into the design of HBOCs. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1676–1684, 2015     

Polymerized placenta hemoglobin attenuates ischemia/reperfusion injury and restores the nitroso-redox balance in isolated rat heart

Ischemia/reperfusion (I/R) injury mainly caused by oxidative stress plays a major role in cardiac damage. The extent of the I/R injury is also an important factor that determines the function of a transplanted heart. This study first examined whether hemoglobin-based oxygen carriers (HBOCs) could protect isolated rat heart from I/R injury and then elucidated the underlying mechanism. Using the Langendorff model, isolated Sprague–Dawley rat hearts were arrested and stored at 4°C for 8 h and then reperfused for 2 h. Compared with St. Thomas' solution (STS) and rat self blood in STS, polymerized placenta hemoglobin (PolyPHb) in STS greatly improved heart contraction and decreased infarction size. The extent of myocardial apoptosis was also significantly decreased, which was related to reduced iNOS-derived nitric oxide production, increased protein ratio of Bcl-2/Bax, and reduced caspase-3 activity and cleavage level. Furthermore, PolyPHb in STS did not increase malondialdehyde, peroxynitrite, or mitochondrial hydrogen peroxide formation, but greatly elevated superoxide dismutase activity and preserved mitochondrial ATP synthesis, which served to maintain redox homeostasis in I/R heart. In conclusion, our results demonstrate that HBOCs protected isolated heart from I/R injury and this protection was associated with attenuation of NO-mediated myocardial apoptosis and restoration of the nitroso-redox balance.     

Rate of NO scavenging alters effects of recombinant hemoglobin solutions on pulmonary vasoreactivity.

Many hemoglobin-based oxygen carriers (HBOCs) produce systemic and pulmonary hypertension and may increase microvascular permeability as a consequence of nitric oxide (NO) scavenging. In this study, we examined the effects of two recombinant human hemoglobin solutions, rHb1.1 and rHb2.0 for injection (rHb2.0), with different rates of NO scavenging on vasoconstrictor reactivity and vascular permeability in isolated, saline-perfused rat lungs. We hypothesized that rHb1.1, a first-generation HBOC with an NO scavenging rate similar to that of native human hemoglobin, would exacerbate pulmonary vasoconstriction and permeability and that rHb2.0, a second-generation HBOC with an NO scavenging rate approximately 20- to 30-fold lower than that of rHb1.1, would minimally influence these responses. Consistent with this hypothesis, rHb1.1 enhanced pulmonary vasoconstrictor reactivity to both hypoxia and thromboxane mimetic U-46619 in a dose-dependent fashion. In contrast, rHb2.0 produced little or no change in reactivity to these stimuli. Furthermore, whereas rHb1.1 abrogated pulmonary vasodilation to the NO-donor S-nitroso-N-acetyl-penicillamine (SNAP), dose-dependent responses to SNAP were preserved, albeit attenuated, in lungs treated with rHb2.0. Finally, the capillary filtration coefficient was unaltered by either rHb1.1 or rHb2.0. We conclude that pulmonary hemodynamic responses to rHb2.0 are greatly reduced compared with those observed with rHb1.1, consistent with rHb2.0 having a diminished capacity to scavenge NO. In addition, neither hemoglobin solution measurably altered microvascular permeability in this preparation.     

血红蛋白类氧载体研究现状

介绍了目前比较有代表性的几类血红蛋白类氧载体的研究现状,着重介绍了第一代血红蛋白类氧载体存在的氧化性损伤问题和第二代偶联抗氧化酶的血红蛋白类氧载体的发展前景。     

Targeted oxygen delivery within hepatic hollow fiber bioreactors via supplementation of hemoglobin-based oxygen carriers

Hepatic hollow fiber bioreactors are considered a promising class of bioartificial liver assist device (BLAD). Unfortunately, limited oxygen (O(2)) transport to hepatocytes within this device hinders further development. Hepatocytes in vivo (in the liver sinusoid) experience a wide range of oxygen tensions (pO(2) = 25-70 mmHg), which is important for development of proper differentiated function (zonation). Previously, we observed that bovine red blood cell (bRBC) supplementation of the circulating media stream enhanced oxygenation of cultured C3A hepatoma cells compared to a culture with no O(2) carrier (Gordon, J.; Palmer, A. F. Artif. Cells, BloodSubstitutes, Biotechnol. 2006, 33 (3), 297-306). Despite this success, the cells were not exposed to the desired in vivo O(2) spectrum (Sullivan, J.; Gordon, J.; Palmer, A. Biotechnol. Bioeng. 2006, 93 (2) 306-317). We hypothesize that altering the kinetics of O(2) binding/release to/from hemoglobin-based O(2) carriers (HBOCs) could potentially target O(2) delivery to cell cultures. High P(50) (low O(2) affinity) HBOCs preferentially targeted O(2) delivery at high inlet pO(2) values. Conversely, low P(50) (high O(2) affinity) HBOCs targeted O(2) delivery at low inlet pO(2) values. Additionally, inlet pO(2), flow rate, and HBOC concentration were varied to find optimal bioreactor operating conditions. Our results demonstrate that HBOCs can enhance O(2) delivery to cultured hepatocytes, while exposing them to in vivo-like O(2) tensions, which is critical to create a fully functional BLAD.     

A compartmental model for oxygen transport in brain microcirculation in the presence of blood substitutes

A compartmental model is developed for oxygen (O(2)) transport in brain microcirculation in the presence of blood substitutes (hemoglobin-based oxygen carriers). The cerebrovascular bed is represented as a series of vascular compartments, on the basis of diameters, surrounded by a tissue compartment. A mixture of red blood cells (RBC) and plasma/extracellular hemoglobin solution flows through the vascular bed from the arterioles through the capillaries to the venules. Oxygen is transported by convection in the vascular compartments and by diffusion in the surrounding tissue where it is utilized. Intravascular resistance and the diffusive loss of oxygen from the arterioles to the tissue are incorporated in the model. The model predicts that most of the O(2) transport occurs at the level of capillaries. Results computed from the present model in the presence of hemoglobin-based oxygen carriers are consistent with those obtained from the earlier validated model (Sharan et al., 1989, 1998a) on oxygen transport in brain circulation in the absence of extracellular hemoglobin. We have found that: (a) precapillary PO(2) gradients increase as PO(2) in the arterial blood increases, P(50 p) (oxygen tension at 50% saturation of hemoglobin with O(2) in plasma) decreases, i.e. O(2) affinity of the extracellular hemoglobin is increased, the flow rate of the mixture decreases, hematocrit decreases at constant flow, metabolic rate increases, and intravascular transport resistance in the arterioles is neglected; (b) precapillary PO(2) gradients are not sensitive to (i) intracapillary transport resistance, (ii) cooperativity (n(p)) of hemoglobin with oxygen in plasma, (iii) hemoglobin concentration in the plasma and (iv) hematocrit when accounting for viscosity variation in the flow; (c) tissue PO(2) is not sensitive to the variation of intravascular transport resistance in the arterioles. We also found that tissue PO(2) is a non-monotonic function of the Hill coefficient n(p) for the extracellular hemoglobin with a maximum occurring when n(p) equals the blood Hill coefficient. The results of the computations give estimates of the magnitudes of the increases in tissue PO(2) as arterial PO(2) increases,P(50 p) increases, flow rate increases, hematocrit increases, hemoglobin concentration in the plasma increases, metabolic rate decreases, the capillary mass transfer coefficient increases or the intracapillary transport resistance decreases.     

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

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

Mixtures of hemoglobin‐based oxygen carriers and perfluorocarbons exhibit a synergistic effect in oxygenating hepatic hollow fiber bioreactors

Hepatic hollow fiber (HF) bioreactors are being developed for use as bioartificial liver assist devices (BLADs). In general, BLADs suffer from O₂ limited transport, which reduces their performance. This modeling study seeks to investigate if O₂ carrying solutions consisting of mixtures of hemoglobin‐based oxygen carriers (HBOCs) and perfluorocarbons (PFCs) can enhance O₂ transport to hepatocytes cultured in the extra capillary space (ECS) of HF bioreactors. We simulated supplementing the circulating cell culture media stream of the HF bioreactor with a mixture containing these two types of oxygen carriers (HBOCs and PFCs). A mathematical model was developed based on the dimensions and physical characteristics of a commercial HF bioreactor. The resulting set of partial differential equations, which describes fluid transport; as well as, mass transport of dissolved O₂ in the pseudo‐homogeneous PFC/water phase and oxygenated HBOC, was solved to yield the O₂ concentration field in the three HF domains (lumen, membrane and ECS). Our results show that mixtures of HBOC and PFC display a synergistic effect in oxygenating the ECS. Therefore, the presence of both HBOC and PFC in the circulating cell culture media dramatically improves transport of O₂ to cultured hepatocytes. Moreover, the in vivo O₂ spectrum in a liver sinusoid can be recapitulated by supplementing the HF bioreactor with a mixture of HBOCs and PFCs at an inlet pO₂ of 80 mmHg. Therefore, we expect that PFC‐based oxygen carriers will be more efficient at transporting O₂ at higher O₂ levels (e.g., at an inlet pO₂ of 760 mmHg, which corresponds to pure O₂ in equilibrium with aqueous cell culture media at 1 atm). Biotechnol. Bioeng. 2010; 105: 534–542. © 2009 Wiley Periodicals, Inc.     

Effect of Cl- and H+ on the oxygen binding properties of glutaraldehyde-polymerized bovine hemoglobin-based blood substitutes

Bovine hemoglobin was cross-linked with glutaraldehyde, resulting in high oxygen affinity polymeric hemoglobin dispersions of varying molecular weight distributions. High oxygen affinity acellular oxygen carriers were designed in order to exhibit oxygen release profiles closer to that of human red blood cells (RBCs), without exhibiting the inherent increased vasoactivity that occurs with low oxygen affinity acellular oxygen carriers (1, 2). Oxygen dissociation curves were measured for polymerized hemoglobin dispersions at various pH values (7.0, 7.4, and 8.0) and chloride ion concentrations. Unmodified hemoglobin showed an increase in oxygen affinity with increased chloride ion concentration and a decrease in oxygen affinity with increased pH, as was previously demonstrated in the literature (3). For glutaraldehyde-polymerized hemoglobin dispersions, the ability of the oxygen affinity to respond to changes in Bohr H+ and Cl- concentration was weakened. However, at acidic physiological pH (pH = 7), the Bohr effect was still present at high Cl- concentrations. Thus, the Bohr effect maintained some dependency on the Cl- concentration.     

Synthesis, biophysical properties, and oxygenation potential of variable molecular weight glutaraldehyde-polymerized bovine hemoglobins with low and high oxygen affinity

In a recent study, ultrahigh molecular weight (Mw ) glutaraldehyde-polymerized bovine hemoglobins (PolybHbs) were synthesized with low O2 affinity and exhibited no vasoactivity and a slight degree of hypertension in a 10% top-load model.(1) In this work, we systematically investigated the effect of varying the glutaraldehyde to hemoglobin (G:Hb) molar ratio on the biophysical properties of PolybHb polymerized in either the low or high O2 affinity state. Our results showed that the Mw of the resulting PolybHbs increased with increasing G:Hb molar ratio. For low O2 affinity PolybHbs, increasing the G:Hb molar ratio reduced the O2 affinity and CO association rate constants in comparison to bovine hemoglobin (bHb). In contrast for high O2 affinity PolybHbs, increasing the G:Hb molar ratio led to increased O2 affinity and significantly increased the CO association rate constants compared to unmodified bHb and low O2 affinity PolybHbs. The methemoglobin level and NO dioxygenation rate constants were insensitive to the G:Hb molar ratio. However, all PolybHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G:Hb molar ratio. In contrast, the colloid osmotic pressure of PolybHbs decreased with increasing G:Hb molar ratio. To preliminarily evaluate the ability of low and high O2 affinity PolybHbs to potentially oxygenate tissues in vivo, an O2 transport model was used to simulate O2 transport in a hepatic hollow fiber (HF) bioreactor. It was observed that low O2 affinity PolybHbs oxygenated the bioreactor better than high O2 affinity PolybHbs. This result points to the suitability of low O2 affinity PolybHbs for use in tissue engineering and transfusion medicine. Taken together, our results show the quantitative effect of varying the oxygen saturation of bHb and G:Hb molar ratio on the biophysical properties of PolybHbs and their ability to oxygenate a hepatic HF bioreactor. We suggest that the information gained from this study can be used to guide the design of the next generation of hemoglobin-based oxygen carriers (HBOCs) for use in tissue engineering and transfusion medicine applications.