Hb-based Oxygen Carriers Research Articles

Rapid reprogramming of haemoglobin structure-function exposes multiple dual-antimicrobial potencies

The intrinsic cytotoxicity of cell-free haemoglobin (Hb) has hampered the development of reliable Hb-based blood substitutes for over seven decades. Notably, recent evidence shows that the Hb deploys this cytotoxic attack against invading microbes, albeit, through an unknown mechanism. Here, we unraveled a rapid molecular reprogramming of the Hb structure-function triggered by virulent haemolytic pathogens that feed on the haem-iron. On direct contact with the microbe, the Hb unveils its latent antimicrobial potency, where multiple antimicrobial fragments are released, each harbouring coordinated 'dual-action centres': microbe binding and pseudoperoxidase (POX) cycle activity. The activated Hb fragments anchor onto the microbe while the juxtaposed POX instantly unleashes a localized oxidative shock, killing the pathogen-in-proximity. This concurrent action conceivably restricts the diffusion of free radicals. Furthermore, the host astutely protects itself from self-cytotoxicity by simultaneously releasing endogenous antioxidants. We found that this decryption mechanism of antimicrobial potency is conserved in the ancient invertebrate respiratory protein, indicating its fundamental significance. Our definition of dual-antimicrobial centres in the Hb provides vital clues for designing a safer Hb-based oxygen carrier blood substitute.

Effects of the molecular mass of tense-state polymerized bovine hemoglobin on blood pressure and vasoconstriction.

Despite recent advances in the design of hemoglobin (Hb)-based oxygen carriers (HBOCs), vasoconstriction, presumably caused by nitric oxide (NO) scavenging, vessel wall hyperoxygenation, and/or extravasation, has been identified as the principal road block hampering commercial development of HBOCs. This study was designed to analyze systemic and microvascular responses to the molecular mass and plasma concentration of tense (T)-state polymerized bovine Hb (PolybHb) solutions. Experiments were performed using the hamster window chamber model subjected to successive hypervolemic infusions of T-state PolybHb solutions. PolybHb plasma concentrations were evaluated, namely, 0.5, 1.0 and 1.5 g/dl, respectively. Infusion of PolybHb solutions with molecular mass >500 kDa elicited hypertension and vasoconstriction proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. However, two high-molecular mass PolybHb solutions, PolybHb(40:1)(high) PolybHb(50:1)(high), did not elicit vasoconstriction at all concentrations studied, whereas PolybHb(50:1)(high) only elicited moderate hypertension at the highest concentration studied. In contrast, infusion of PolybHb solutions with molecular mass <500 kDa elicited significant hypertension and vasoconstriction compared with PolybHb solutions with molecular mass >500 kDa that was proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. We present promising results for highly cross-linked T-state PolybHb solutions with molecular mass >500 kDa [PolybHb(40:1)(high) PolybHb(50:1)(high)], which supports the concept that HBOC size/molecular mass influences its proximity to the vascular endothelium and molecular diffusivity. The hemodynamics of HBOC within the plasma layer surrounding the abluminal side endothelium regulates NO production and consumption, vessel oxygen flux, and extravasation. Although mechanistically attractive, neither of these hypotheses can be directly tested in vivo and will require further investigation.

Mixed S-Nitrosylated Polymerized Bovine Hemoglobin Species Moderate Hemodynamic Effects in Acutely Hypoxic Rats

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being developed as a potential therapy for increasing tissue oxygenation, yet they have not reached their full potential because of unwanted hemodynamic side effects (vasoconstriction, low cardiac output, and oxygen delivery) due in part to nitric oxide (NO) scavenging by cell-free Hb. It may be possible to overcome the NO scavenging effect by coinfusing S-nitrosylated (SNO) HBOC along with unmodified HBOC. SNO-HBOC, like free Hb, may act as an NO donor in low-oxygen conditions. We hypothesized that an unaltered HBOC, polymerized bovine Hb (PBvHb), coinfused with an SNO-PBvHb, would improve hemodynamics and oxygen delivery during hypoxia. Vascular oxygen content and hemodynamics were determined after euvolemic rats were infused (3 ml) with lactated Ringer's solution, PBvHb, SNO-PBvHb, or PBvHb plus SNO-PBvHb (1:10) during normoxia or acute hypoxia (fraction of inspired oxygen = 10%, 120 min). Hemodynamic side effects resulting from PBvHb infusion (vasoconstriction, elevated pulmonary blood pressure, and reduced cardiac output) were offset by SNO-PBvHb in acute hypoxic, but not normoxic, conditions. These data support the potential use of HBOC mixed with SNO-HBOC for the treatment of conditions in which acute hypoxia is present, such as tumor oxygenation, wound healing, hemorrhagic trauma, and sickle cell and hemolytic anemia.

Attenuated erythrocytic ATP release is caused by extracellular hemoglobin.

Local regulation of microvascular flow is a complex process evolved to match oxygen supply to the metabolic demands of the tissue. The RBC has been proposed as an O2 sensor through a direct link between the desaturation of intracellular hemoglobin (Hb) and ATP release, leading to vasodilation1. This theory suggests a link between alterations of O2 transport parameters in the plasma space and vasoactivity. Based on theoretical calculations of O2 extraction from RBCs in the presence of Hb‐based oxygen carriers (HBOCs), we hypothesized that that the addition of cell‐free Hb to the extracellular space provides a supplementary O2 source that reduces RBC desaturation and ATP release. In this study, the total O2 concentration of RBC/HBOC suspensions was lowered by additions of deoxygenated saline, and then assayed for ATP production via the luciferin‐luciferase bioluminescence assay. When an acellular Hb cross‐linked between α subunits (αα Hb, p50 = 33 mmHg) was added to the red cell suspension, ATP production was significantly less in the absence of HBOC. These results provide a possible mechanism for HBOC‐induced vasoactivity through delayed release of red cell ATP.1 Ellsworth, ML (2000) Acta Physiol Scand 168: 551‐559.

Increased arterial oxygen content by artificial haemoglobin induces a decrease in regional cerebral blood flow and decreased regional cerebral oxygen delivery

Under physiological conditions, cerebral oxygen delivery is kept constant by adaptation of the regional cerebral blood flow (CBF) in relation to the oxygen content. So far, decreases of the regional CBF induced by a higher arterial oxygen content have been produced under hyperbaric or hyperviscous conditions. We tested whether local CBF is also reduced by a high haemoglobin (Hb) concentration at a normal haematocrit (Hct). Compared with controls (n=8), Hb content was increased to 19 g dl(-1) in conscious rats by isovolaemic replacement of the plasma fraction with an artificially high Hb solution (Hb-based oxygen carriers; HH group, n=8). In another group (n=8), Hct was decreased by isovolaemic exchange with an Hb-based oxygen carrier resulting in a normal Hb content (NH group). Mean and regional CBF was measured by iodo-[(14)C]-antipyrine autoradiography. Oxygen delivery was calculated from arterial oxygen content and CBF. Compared with the controls (Hb 15.3 g dl(-1), Hct 0.44), mean CBF was lower in the HH (Hb 20.3 g dl(-1), Hct 0.44) group by 23% (P < or = 0.05), but remained unchanged in the NH group (Hb 15.0 g dl(-1), Hct 0.29). On a local level, hyperoxygenation reduced CBF in 22 out of 39 brain regions. In the NH group mean CBF was unchanged, whereas local CBF was higher in 10 areas. In both groups, overall cerebral oxygen delivery was unchanged compared with the control group. Locally though, high arterial Hb content decreased oxygen delivery in one-third of the brain structures. Whereas the overall cerebral oxygen delivery in the brain is maintained during hyperoxygenation and haemodilution, local oxygen delivery is decreased by high arterial Hb content in some brain regions.

Review of Hemoglobin‐Vesicles as Artificial Oxygen Carriers

Blood transfusion systems have greatly benefited human health and welfare. Nevertheless, some problems remain: infection, blood type mismatching, immunological response, short shelf life, and screening test costs. Blood substitutes have been under development for decades to overcome such problems. Plasma component substitutes have already been established: plasma expanders, electrolytes, and recombinant coagulant factors. Herein, we focus on the development of red blood cell (RBC) substitutes. Side effects hindered early development of cell-free hemoglobin (Hb)-based oxygen carriers (HBOCs) and underscored the physiological importance of the cellular structure of RBCs. Well-designed artificial oxygen carriers that meet requisite criteria are expected to be realized eventually. Encapsulation of Hb is one idea to shield the toxicities of molecular Hbs. However, intrinsic issues of encapsulated Hbs must be resolved: difficulties related to regulating the molecular assembly, and management of its physicochemical and biochemical properties. Hb-vesicles (HbV) are a cellular type of HBOC that overcome these issues. The in vivo safety and efficacy of HbV have been studied extensively. The results illustrate the potential of HbV as a transfusion alternative and promise its use for other clinical applications that remain unattainable using RBC transfusion.

Hemoglobin Conjugated with a Band 3 N-terminus Derived Peptide as an Oxygen Carrier

A peptide composed of 9 amino acids, 7 residues from N-terminus of human erythrocytic Band 3 protein (AcMEELQDD) followed by cysteine and glutamic acids, was conjugated to hemoglobin (Hb) serving as an allosteric effector for oxygen release. The activated polyethylene glycol (PEG), maleimide-PEG-N-hydroxysuccinimidyl, was used to crosslink Hb with the peptide. The putative conjugation site on Hb for effective enhancement of oxygen release was characterized as Lys-beta95 by liquid chromatography-tandem mass spectrometry. In addition, the conjugated peptide causes a rightward shift of the oxygen dissociation curve as compared to that of its parent Hb when the degree of oxygen saturation is higher than 50%. Furthermore, this conjugated peptide remains effective on lowering Hb's oxygen affinity after Hb polymerization by another PEG crosslinker. The allosteric properties of the peptide-conjugated Hb may provide a new aspect of Hb-based oxygen carriers.

Tangential flow filtration of hemoglobin

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P(50)) and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (alpha) and beta (beta) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the alpha and beta globin chains agreed well with the calculated theoretical mass of the alpha- and beta- globin chains. Taken together, our results demonstrate that HPLC-grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb-based oxygen carriers.

Site-Selective Glycosylation of Hemoglobin on Cys β93

In this work, we describe the synthesis and characterization of a novel glycosylated hemoglobin (Hb) with high oxygen affinity as a potential Hb-based oxygen carrier. Site-selective glycosylation of bovine Hb was achieved by conjugating a lactose derivative to Cys 93 on the beta subunit of Hb. LC-MS analysis indicates that the reaction was quantitative, with no unmodified Hb present in the reaction product. The glycosylation site was identified by chymotrypsin digestion of the glycosylated bovine Hb followed with LC-MS/MS and from the X-ray crystal structure of the glycosylated Hb. The chemical conjugation of the lactose derivative at Cys beta93 yields an oxygen carrier with a high oxygen affinity (P(50) of 4.94 mmHg) and low cooperativity coefficient (n) of 1.20. Asymmetric flow field-flow fractionation (AFFFF) coupled with multiangle static light scattering (MASLS) was used to measure the absolute molecular weight of the glycosylated Hb. AFFFF-MASLS analysis indicates that glycosylation of Hb significantly altered the alpha(2)beta(2)-alphabeta equilibrium compared to native Hb. Subsequent X-ray analysis of the glycosylated Hb crystal showed that the covalently linked lactose derivative is sandwiched between the beta(1) and alpha(2) (and hence by symmetry the beta(2) and alpha(1)) subunits of the tetramer, and the interaction between the saccharide and amino acid residues located at the interface is apparently stabilized by hydrogen bonding interactions. The resultant structural analysis of the glycosylated Hb helps to explain the shift in the alpha(2)beta(2)-alphabeta equilibrium in terms of the hydrogen bonding interactions at the beta(1)alpha(2)/beta(2)alpha(1) interface. Taken together, all of these results indicate that it is feasible to site-specifically glycosylate Hb. This work has great potential in developing an oxygen carrier with defined chemistry that can target oxygen delivery to low pO(2) tissues and organs.

An overview of unresolved inherent problems associated with red cell transfusion and potential use of artificial oxygen carriers and ECO-RBC: Current status/future trends

This manuscript deals with why we need alternatives to liquid stored RBC highlighting some of the unresolved inherent problems related to red cell storage lesion and their potential impacts on the clinical outcomes and transfusion complications. The promise of several potential alternatives to red cell transfusions such as: Perfluorocarbon; Modified Hb-based oxygen carriers and newer design of Hb-based oxygen carriers are reviewed. It is noteworthy to say that since the first introduction of these oxygen carriers, almost five decades ago, the only successful drive has been to prepare safer and more convenient oxygen carriers, for enhancing the quality of life of recipients and their usage, either as substitutes to red cell transfusion or even as the bridge, remains patchy. Moreover, as new products with better characteristics become available the older products from the competitors are withdrawn. Finally, the current progress on universal RBC, known as ECO-cells is highlighted and, in the future perspectives, some of the current efforts in making the red cells transfusion safer and more efficacious are briefly addressed.

Cerebral oxygen delivery by liposome-encapsulated hemoglobin: a positron-emission tomographic evaluation in a rat model of hemorrhagic shock

Liposome-encapsulated Hb (LEH) is being developed as an artificially assembled, low-toxicity, and spatially isolated Hb-based oxygen carrier (HBOC). Standard methods of evaluating oxygen carriers are based on surrogate indicators of physiology in animal models of shock. Assessment of actual delivery of oxygen by HBOCs and resultant improvement in oxygen metabolism at the tissue level has been a technical challenge. In this work, we report our findings from 15O-positron emission tomographic (15O-PET) evaluation of LEH in a rat model of 40% hypovolemic shock. In vitro studies showed that PEGylated LEH formulation containing approximately 7.5% Hb and consisting of neutral lipids (distearoylphosphatidylcholine:cholesterol:alpha-tocopherol, 51.4:46.4:2.2) efficiently picks up 15O-labeled oxygen gas. The final preparation of LEH contained 5% human serum albumin to provide oncotic pressure. Cerebral PET images of anesthetized rats inhaling 15O-labeled O2 gas showed efficient oxygen-carrying and delivery capacity of LEH formulation. From the PET images, we determined cerebral metabolic rate of oxygen (CMR(O2)) as a direct indicator of oxygen-carrying capacity of LEH as well as oxygen delivery and metabolism in rat brain. Compared with control fluids [saline and 5% human serum albumin (HSA)], LEH significantly improved CMR(O2) to approximately 80% of baseline level. Saline and HSA resuscitation could not improve hypovolemia-induced decrease in CMR(O2). On the other hand, resuscitation of shed blood was the most efficient in restoring oxygen metabolism. The results suggest that 15O-PET technology can be successfully employed to evaluate potential oxygen carriers and blood substitutes and that LEH resuscitation in hemorrhage enhances oxygen delivery to the cerebral tissue and improves oxygen metabolism in brain.

Gating the Radical Hemoglobin to Macrophages: The Anti-Inflammatory Role of CD163, a Scavenger Receptor

Efficient extracellular hemoglobin (Hb) clearance is essential to prevent oxidative- and nitrosative-mediated toxicity. CD163 belongs to group B of the scavenger receptor cysteine-rich (SRCR) protein family found on the surface of monocytes and macrophages and is responsible for Hb-haptoglobin (Hp) complex uptake. Hb uptake by CD163 was thought to proceed exclusively through an Hp-dependent pathway. However, Hb can interact directly with CD163 via a low affinity binding when Hp is absent. As a result, a two-phase hypothesis of Hb clearance by monocytes/macrophages suggests that Hp-Hb binding to CD163 is the primary mechanism of plasma Hb clearance, while clearance of Hb by direct binding to CD163 is secondary to Hp depletion. The authors have considered the ligand specificity of CD163 in human macrophages and in a heterologous gene expression model to demonstrate that Hb is effectively endocytosed by CD163 in the absence of Hp. Additionally, the authors have considered Hb-based oxygen carriers (HBOCs) administration as a unique situation during which direct CD163 uptake may be relevant as a mechanism of clearance. However, the nature of chemical modifications introduced onto the Hb molecule and/or oxidative changes induced in the protein appear to influence the extent of CD163 interaction and cellular uptake. Here, an overview and novel insights into the role of CD163 in Hb redox inactivation and clearance are provided.

First-generation blood substitutes: what have we learned? Biochemical and physiological perspectives

Chemically modified or recombinant hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as oxygen therapeutics or ‘blood substitutes’ for use in a variety of clinical settings. Oxidative and nitrosative reactions of acellular Hb can limit the effectiveness and compromise the safety of HBOCs. The reactions between Hb and biologically relevant redox active molecules may also perturb redox sensitive signaling pathways. In recent years, systematic in vitro and in vivo structural and functional evaluation of several HBOCs has been carried out and, in some cases, delineated the ‘structural’ origin of their toxicity. This enables potential protective strategies against Hb-mediated side reactions to be rationally suggested. Here the authors provide an overview of their research experiences, novel insights into the molecular basis of toxicities of these products and some lessons learned.

Coagulation patterns following haemoglobin‐based oxygen carrier resuscitation in severe uncontrolled haemorrhagic shock in swine

Massive blood loss due to penetrating trauma and internal organ damage can cause severe haemorrhagic shock (HS), leading to a severely compromised haemostatic balance. This study evaluated the effect of bovine polymerized haemoglobin (Hb) (Hb-based oxygen carrier, HBOC) resuscitation on haemostasis in a swine model of uncontrolled HS. Following liver injury/HS, swine received HBOC (n= 8), Hextend (HEX) (n= 8) or no resuscitation (NON) (n= 8). Fluids were infused to increase mean arterial pressure above 60 mmHg and to reduce heart rate to baseline. At 4 h, the animals were eligible for blood transfusions. Prothrombin time (PT), activated partial thromboplastin time, fibrinogen, thromboelastography (TEG) and platelet function analyser closure time (PFA-CT) were compared by using mixed statistical model. At 4 h, blood loss (% estimated blood volume) was comparable for HBOC (65.5 +/- 18.5%) and HEX (80.8 +/- 14.4%) and less for NON (58.7 +/- 10.1%; P < 0.05). Resuscitation-induced dilutional coagulopathy was observed with HBOC and HEX, as indicated by reduced haematocrit, platelets and fibrinogen (P < 0.05). At 4 h, PT was higher in HEX than in HBOC groups (P < 0.01). In the early hospital phase, a trend to increased TEG reaction time and PFA-CT indicates that dilutional effects persist in HBOC and HEX groups. PFA-CT returned to baseline later with HBOC than with HEX (48 vs. 24 h) following blood transfusion. At 4 h, all surviving HEX animals (n= 3) required transfusion, in contrast to no HBOC (n= 7) or NON (n= 1) animals. In this severe uncontrolled HS model, successful resuscitation with HBOC produced haemodilutional coagulopathy less than or similar to that produced by resuscitation with HEX.

Chemical Characterization of Diaspirin Cross-Linked Hemoglobin Polymerized with Poly(ethylene glycol)

A lack of specificity associated with chemical modification methods used in the preparation of certain hemoglobin (Hb)-based oxygen carriers (HBOCs) may alter Hb structure and function, as amino acids located in critical regions (e.g., alpha-beta interfaces and the 2,3-DPG binding pocket) may unintentionally be targeted. Hb protein surface modifications with various poly(ethylene glycol) (PEG) derivatives have been used as conjugating and polymerizing agents with the intent of improving reaction site specificity/reproducibility and ultimately reducing the untoward hypertensive response due to nitric oxide scavenging by smaller molecular size tetrameric species (i.e., 64 kDa) in HBOC solutions. Previous experiments performed in our laboratory have evaluated the influence of polymerization of diaspirin alpha-alpha cross-linked Hb (alphaalpha-DBBF-Hb) with a bifunctional modified PEG, bis(maleoylglycylamide) PEG (BMAA-PEG), in terms of oxygen carrying capacity, redox properties, hypertensive response, and renal clearance in rats. The data presented in this paper specifically evaluate the influence of BMAA-PEG on alphaalpha-DBBF-Hb (Poly-alphaalpha-DBBF-Hb) to identify molecular weight distribution, protein conformation, and site-specific modification, as well as to provide insight into the previously determined in vitro and in vivo functional and vasoactive characteristics of this HBOC. Chemical analysis performed herein reveals nonspecific modifications induced by BMAA-PEG that result in the full modification of alphaalpha-DBBF-Hb leaving no tetrameric cross-linked starting material in solution. These data are inconsistent with the continuing assumption that molecular size (i.e., 64 kDa) has a direct influence on HBOC-mediated vasoactivity and that other protective strategies should be considered to control blood pressure imbalances.

Synthesis and characterization of a novel DTPA polymerized hemoglobin based oxygen carrier

The purpose of this study was to prepare a novel polymerized hemoglobin (Hb) based oxygen carrier (HBOC) designed to minimize Hb induced hypertension, while employing a simple and inexpensive method of preparation. Cyclic-diethylenetriaminepentaacetic acid (DTPA) was used to polymerize stroma free Hb (SF-Hb). SF-Hb was isolated from red blood cells and reacted with DTPA at a constant concentration, pH, and duration. Low molar mass fractions (<100 kDa) were removed using ultrafiltration. Reactions and subsequent ultrafiltration steps were determined to be reproducible by analyzing molar mass, colloid osmotic pressure and oxygen affinity. Finally, a model of 50% exchange transfusion (ET) in rats was used to evaluate the blood pressure response to DTPA polymerized SF-Hb (Poly-DTPA-Hb). Poly-DTPA-Hb demonstrated a number averaged molar mass of 128.7 kDa and a weighted average of 223.0 kDa. Oxygen binding equilibrium indicated high oxygen affinity (P50 = 5.1+/-0.01 mm Hg) and little cooperativity (n = 1.4). Poly-DTPA-Hb and a control DTPA polymerized human serum albumin (Poly-DTPA-HSA) unexpectedly caused acute hypotension during the period of ET in rats (mean arterial pressure approximately 45% less than baseline). Hypotension occurring over the period of ET was determined to be mediated by calcium binding to protein associated DTPA. This effect was attenuated by the addition of calcium chloride (CaCl2) to the Poly-DTPA protein preparations. Cyclic DTPA anhydride can be used to create cross-linked and polymerized hemoglobin, using a simple and inexpensive process. However, the addition of CaCl2 to the preparation appears to be required to prevent calcium chelation and subsequent hypotension during infusion.

The Pharmacokinetics of Hemoglobin-Based Oxygen Carrier Hemoglobin Glutamer-200 Bovine in the Horse

Hemoglobin-glutamer-200 (HBOC-200) is a hemoglobin (Hb)-based oxygen carrier (HBOC) comprising glutaraldehyde-polymerized bovine Hb. In this study, we sought to determine the pharmacokinetics of this first generation HBOC after IV infusion of 32.5 g of HBOC-200 solution in horses. Quantification of HBOC-200 in equine plasma and urine was performed using a method recently developed by our laboratory. The elimination from plasma was based on size distribution of the bovine Hb polymer. The decline of plasma concentration-time curve of HBOC-200 was described by a noninterchanging 2-compartmental model. The median elimination half-lives of the small and large aggregates were 1.3 and 12.0 h, respectively. Of the HBOC-200 infused, 47.0% was eliminated as the smaller molecular weight and 53% as the larger molecular weight polymers. The area under the plasma concentration-time curve was 5143.1 microg.h(-1).mL(-1). The volumes of distribution of the small and large aggregates were 86.9 and 63.9 mL/kg and the clearances were 42.1 and 3.8 mL.kg(-1).h(-1), respectively. In conclusion, elimination of first generation HBOCs was shown to be more complex than previously assumed because of the heterogeneous nature of these solutions. Mammalian species dispose of Hb using similar mechanisms, and there is no unique metabolic process in the horse that would not allow a logical extension of the general interpretation of this study.

Systemic Responses to Hemodilution After Transfusion with Stored Blood and with a Hemoglobin-Based Oxygen Carrier

We assessed the systemic effects of exchanges with blood or hemoglobin (Hb) raffimer under conditions of critical oxygen delivery (Do(2)crit). We compared Do(2)crit in animals receiving Hb-based oxygen carrier (HBOC; Hemolink), fresh blood (collected <24 h), or stored blood (10 days) before hemodilution. Rats were randomized to control, blood, or HBOC isovolemic exchange. Oxygen consumption was measured by using expired gas (o(2)a) and blood (o(2)b) samples, whereas whole-body oxygen delivery (Do(2)) was calculated from cardiac output and arterial oxygen content. After exchange, rats were subjected to stepwise isovolemic hemodilution. Blood pressure, gases, acid-base status, glucose, Hb oxygen saturation, heart rate, and total peripheral resistance were also measured. We found that 1) HBOC-treated rats showed an increased mean arterial blood pressure and total peripheral resistance throughout the hemodilution, 2) Do(2)crit calculated with o(2)a or o(2)b gave identical results, 3) Do(2)crit was not different between animals receiving blood and those receiving HBOC, 4) the terminal Hb concentration (1.8 +/- 0.1 g/dL) and Do(2) (5 +/- 1 mL . min(-1) . kg(-1)) were similar for all animals, and 5) most oxygen transport and biochemical variables changed similarly during hemodilution. The data suggest that tolerance to Do(2)crit is not altered by 50% replacement of native Hb by stored blood or Hb raffimer.

Enhanced Molecular Volume of Conservatively Pegylated Hb: (SP-PEG5K)6-HbA is Non-Hypertensive

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.

Large volume polymerized haemoglobin solution in a Jehovah's Witness following abruptio placentae.

Severe anaemia, with haemoglobin (Hb) levels < or =3 g dL(-1), is associated with mortality rates of 50-95%. Although accepted transfusion targets have been debated in the literature (Carson et al., 2002; Practice guidelines for blood component therapy. 1996; Consensus Conference. 1988; Hebert et al., 1999), few would argue the risks associated with Hb levels less than 5 g dL(-1) in critically ill patients. In patients who are unable to receive red blood cell transfusions, the utility of Hb solutions is an attractive solution. We describe a Jehovah's Witness patient who exemplifies the marked physiologic derangements of severe anaemia and subsequent clinical resolution with large volume polymerized human Hb transfusion. The Hb-based oxygen carrier, PolyHeme, provided adequate oxygen transport, acting as a bridge until endogenous production could compensate for red cell loss. Practicing physicians need to be aware of current therapeutic options for use in these complicated patients.