Cell-free Hemoglobin Solutions Research Articles

Scalable manufacturing platform for the production of methemoglobin as a non-oxygen carrying control material in studies of cell-free hemoglobin solutions.

Methemoglobin (metHb) arises from the oxidation of ferrous hemoglobin (HbFe2+, Hb) to ferric hemoglobin (HbFe3+, metHb), which is unable to bind gaseous ligands such as oxygen (O2) and carbon monoxide (CO), and binds to nitric oxide (NO) significantly slower compared to Hb. Therefore, metHb does not elicit vasoconstriction and systemic hypertension in vivo due to its extremely slow NO scavenging rate in comparison to cell-free Hb, but will induce oxidative tissue injury, demonstrating the potential of using metHb as a control material when studying the toxicity of cell-free Hb. Hence, the goal of this work was to develop a novel manufacturing strategy for production of metHb that is amenable to scale-up. In this study, small scale (e.g. 1 mL reaction volume) screening experiments were initially conducted to determine the optimal molar ratio of Hb to the oxidization agents hydrogen peroxide (H2O2) or sodium nitrite (NaNO2) to achieve the highest conversion of Hb into metHb. A spectral deconvolution program was employed to determine the molar fraction of various species (hemichrome, metHb, oxyHb, metHb-, and NaNO2) in solution during the oxidation reaction. From this analysis, either a 1:1 or 1:5 molar ratio was identified as optimal molar ratios of Hb:NaNO2 (heme basis) that yielded the highest conversion of Hb into metHb with negligible amounts of side products. Hence in order to reduce the reaction time, a 1:5 molar ratio was chosen for large scale (i.e. 1.5 L reaction volume) synthesis of bovine metHb (metbHb) and human metHb (methHb). The biophysical properties of metHb were then characterized to elucidate the potential of using the synthesized metHb as a non-O2 carrying control material. The haptoglobin binding kinetics of metHb were found to be similar to Hb. Additionally, the synthesized metHb was stable in phosphate buffered saline (PBS, 50 mM, pH 7.4) at 4°C for approximately one week, indicating the high stability of the material.

Hemoglobin oxygen fractional saturation regulates nitrite-dependent vasodilation of aortic ring bioassays

Nitrite reacts with deoxyhemoglobin to generate nitric oxide (NO). This reaction has been proposed to contribute to nitrite-dependent vasodilation in vivo and potentially regulate physiological hypoxic vasodilation. Paradoxically, while deoxyhemoglobin can generate NO via nitrite reduction, both oxyhemoglobin and deoxyhemoglobin potently scavenge NO. Furthermore, at the very low O(2) tensions required to deoxygenate cell-free hemoglobin solutions in aortic ring bioassays, surprisingly low doses of nitrite can be reduced to NO directly by the blood vessel, independent of the presence of hemoglobin; this makes assessments of the role of hemoglobin in the bioactivation of nitrite difficult to characterize in these systems. Therefore, to study the O(2) dependence and ability of deoxhemoglobin to generate vasodilatory NO from nitrite, we performed full factorial experiments of oxyhemoglobin, deoxyhemoglobin, and nitrite and found a highly significant interaction between hemoglobin deoxygenation and nitrite-dependent vasodilation (P < or = 0.0002). Furthermore, we compared the effect of hemoglobin oxygenation on authentic NO-dependent vasodilation using a NONOate NO donor and found that there was no such interaction, i.e., both oxyhemoglobin and deoxyhemoglobin inhibited NO-mediated vasodilation. Finally, we showed that another NO scavenger, 2-carboxyphenyl-4,4-5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibits nitrite-dependent vasodilation under normoxia and hypoxia, illustrating the uniqueness of the interaction of nitrite with deoxyhemoglobin. While both oxyhemoglobin and deoxyhemoglobin potently inhibit NO, deoxyhemoglobin exhibits unique functional duality as an NO scavenger and nitrite-dependent NO generator, suggesting a model in which intravascular NO homeostasis is regulated by a balance between NO scavenging and NO generation that is dynamically regulated by hemoglobin's O(2) fractional saturation and allosteric nitrite reductase activity.

Blood oxyhemoglobin saturation measurements by blue-green spectral shift

Previous work describing a resilient method for measuring oxyhemoglobin saturation using the blue-green spectral shift was performed using cell free hemoglobin solutions. Hemoglobin solution and whole blood sample spectra measured under similar conditions in a spectrophotometer are used here to begin evaluating the impact of cellular scattering on this method. The blue-green spectral shift with changing oxyhemoglobin saturation was preserved in these blood samples and the blue-green spectral shift was relatively unaffected by physiological changes in blood pH (6.6, 7.1, and 7.4), path length through blood (100 and 200 microm), and blood hematocrit (19 to 48%). The packaging of hemoglobin in red blood cells leads to a decreased apparent path length through hemoglobin, and an overall decrease in scattering loss with increasing wavelength from 450 to 850 nm. The negative slope of the scattering loss in the 476 to 516-nm range leads to a +3.0 nm shift in the oxyhemoglobin saturation calibration line when the blue-green spectral minimum in these blood samples was compared to cell free hemoglobin. Further research is needed to fully evaluate the blue green spectral shift method in cellular systems including in vivo testing.

Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse

Cell-free hemoglobin solutions with high oxygen affinity might be beneficial for selectively delivering oxygen to ischemic tissue. A recombinant hybrid hemoglobin molecule was designed using the human alpha-subunit and the bovine beta-subunit, with placement of surface cysteines to permit disulfide bond polymerization of the tetramers. The resulting protein generated from an Escherichia coli expression system had a molecular mass >1 MDa, a P50 of approximately 3 Torr, and a cooperativity of n = 1.0. Anesthetized mice were transfused during 2-h occlusion of the middle cerebral artery. Compared with transfusion with 5% albumin, cerebral infarct volume was reduced by 41% with transfusion of a 3% solution of the high oxygen-affinity hemoglobin polymer and by 50% with transfusion of a 6% solution of the polymer. Transfusion of a 6% solution of a 500-kDa polymer possessing a P50 of 17 Torr and a cooperativity of n = 2.0 resulted in a 66% reduction of infarct volume. These results indicate that cell-free Hb polymers with P50 values much lower than that of red blood cell hemoglobin are highly capable of salvaging ischemic brain. The assumption that the P50 of blood substitutes should be similar to that of blood might not be warranted when used during ischemic conditions.

Use of the ADVIA 120 for differentiating extracellular and intracellular hemoglobin

The use of cell-free hemoglobin (Hgb) solutions, such as Oxyglobin (Biopure Corp, Cambridge, MA, USA), as a blood substitute for the treatment of acute anemias is increasing in veterinary medicine. These solutions interfere with colorimetric tests, which do not discriminate between cellular Hgb (Hgb-cell) from the patient and extracellular Hgb (Hgb-delta) from the Oxyglobin, and therefore make the monitoring of anemia, based on Hgb concentration, difficult. The ADVIA 120 hematology analyzer (Bayer Diagnostics, Tarrytown, NY, USA) evaluates Hgb by 2 methods, a standard cyanmethemoglobin colorimetric method and flow cytometry, and therefore might provide the means to differentiate extracellular and intracellular Hgb. The objective of this study was to determine the accuracy and precision of the ADVIA 120 in differentiating extracellular from intracellular Hgb. Anticoagulated whole blood samples from 10 healthy dogs were analyzed in triplicate on the ADVIA 120. Hgb-delta concentration was determined by adding Oxyglobin (13 g/dL) to the whole blood samples at dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, and 1:32. Hgb-cell and Hgb-total values were calculated and compared with actual values by linear regression. Analyses were done in triplicate and repeated 9 consecutive times to evaluate intra-assay precision of Hgb-total and Hgb-cell determinations. Correlation between Hgb values obtained by colorimetric (Hb-total) and flow cytometric (Hgb-cell) methods on whole blood samples was high (R(2) = .99; n = 10) with a slope of 0.96 and intercept of 0. Correlation between actual and predicted Hgb-cell values also was high (R(2) = .99), with a small positive bias (0.289 +/- 0.185; n = 60). Intra-assay precisions were high, with most coefficients of variation <2%. The ADVIA 120 is capable of differentiating Hgb-cell from Hgb-delta. The flow cytometric method is accurate and precise when compared with the cyanmethemoglobin method. A small bias between the results is unlikely to be clinically significant but may affect the ability of the ADVIA to differentiate small quantities (<0.3 g/dL) of Hgb-delta.

Alternatives to blood in the 21st century

Persons who suffer traumatic injury are likely to be transfused with considerable amounts of blood during initial resuscitation efforts. Oxygen-carrying solutions are currently in clinical testing as substitutes for red blood cells. Although these agents may eliminate many concerns associated with blood administration (short shelf life, infectious and immunologic risks, the need to type and cross-match), early cell-free hemoglobin solutions demonstrated nephrotoxicity and were associated with pulmonary and systemic hypertension, among other adverse events. Newer polymerized hemoglobin solutions show acceptable safety profiles in the surgical setting and studies are being designed, some with funding from the US Department of Defense, to evaluate their efficacy in hemorrhaging trauma victims.

Hemoglobin solutions.

To review current knowledge about cell-free hemoglobin solutions. A computerized MEDLINE search was used to retrieve all studies concerning cell-free hemoglobin solutions from 1990 to 2003. The reference lists of all available review articles and primary studies were also reviewed to identify references not identified in the computerized search. All clinical and experimental studies involving cell-free hemoglobin solutions were included. From the selected studies, information was obtained regarding the experimental model or the study population in which cell-free hemoglobin solutions were investigated, the type of cell-free hemoglobin solution used, their deleterious or beneficial effects, and their possible indications. In many studies, hemoglobin solutions were considered as efficient resuscitative agents and good alternatives to red blood cell transfusion, owing to their marked vasopressor effect, coupled with their capacity to improve the microcirculation and rapidly restore metabolic parameters. The main problems identified include excessive systemic vasoconstriction and oxidative damage. Initial enthusiasm in the development of hemoglobin solutions has been tempered recently by the negative results of a U.S. multicenter trial studying the early infusion of diaspirin cross-linked hemoglobin in trauma patients. Nevertheless, the properties of diaspirin cross-linked hemoglobin (and particularly the strong vasopressor effects) cannot be attributed to all hemoglobin solutions, and results of new clinical studies are eagerly awaited to evaluate the potential benefit of such solutions in the management of trauma patients. Today, we are aware of the effects of the first generation of blood substitutes. Further research is ongoing into newer solutions. One area of interest is the development of new molecular structures to decrease nitric oxide binding, thus minimizing any adverse events and maximizing potential benefits. Nevertheless, possible adverse effects need to be carefully evaluated before these agents can be widely administered.

Different efficacy in vitro of hemoglobin based oxygen carriers and red cells.

A vast literature exists which either theoretically explores the mechanism of oxygen delivery from blood to the surrounding tissues [1,3], or simulates oxygen delivery and absorption by red cells suspension and cell free hemoglobin solutions using artificial capillary systems [4,6]. Very little is known about the physiologic efficiency of cell free hemoglobin solutions to sustain life of isolated organs and tissues. We thought that the oxygen dependent transport of metabolites across ileal mucosa, superfused in Ussing chambers, could be used to test the efficacy of hemoglobin based oxygen carriers [7].

Current status of cell-free hemoglobin solutions

Current Status of Cell-Free Haemoglobin Solutions. Persisting concern about the risks of allogeneic blood and the impending shortfall of such products have reinforced the search for alternative erythrocyte substitutes in recent years. With the absence of toxic side effects of modern, ultrapurified and chemically modified haemoglobin-based oxygen carriers (HBOC), many studies in animals, volunteers and patients have shown their effectivity and tolerability. HBOC can be applied in patients without prior cross-matching and one product, which is available as room temperature-stable formulation with a shelf-life of > 2 years, has recently been approved for the treatment of peri-operative anaemia in South Africa. While clinical studies with human cross-linked haemoglobin (DCLHb) were discontinued because of an increased mortality of patients receiving DCLHb after stroke and multiple injury shock, phase III studies with polymerized bovine haemoglobin (HBOC-201) have been completed showing that peri-operative infusion of HBOC-201 was able to avoid or reduce allogeneic blood transfusion. Different human, recombinant and bovine hemoglobin solutions are currently under investigation in cardiac surgery, sepsis and tumour patients. Extensive laboratory investigations have shown that HBOC are highly effective oxygen donors with regard to tissue oxygenation rather than erythrocyte substitutes in case of bleeding, because they provide plasmatic oxygen delivery under physiological concentrations of inspired oxygen. HBOC have advantages over red blood cells because of their easier oxygen release per se and from adjacent erythrocytes, and their capability to reach poststenotic or poorly perfused tissues with the plasma stream.

Hemoglobin solutions—Not just red blood cell substitutes

To review current knowledge about cell-free hemoglobin solutions. All studies involving cell-free hemoglobin were retrieved from a computerized MEDLINE search from 1980 to 1998. We also reviewed the reference lists of all available review articles and primary studies to identify references not found in the computerized search. Clinical and experimental studies in which cell-free hemoglobin solutions were studied. From the selected studies, information was obtained regarding the experimental model or the study population in which cell-free hemoglobin solutions were investigated, the type of cell-free hemoglobin solution, their deleterious or beneficial effects, and their possible indications. In many studies, hemoglobin solutions were considered as efficient resuscitative agents and good alternatives to red blood cell transfusion, because of their marked vasopressor effect coupled with their capacity to improve the microcirculation and quickly restore metabolic parameters. Nevertheless, potential problems include an increased susceptibility to infection, immunosuppression, oxidative damage, excessive pulmonary and systemic vasoconstriction, and platelet activation. Hemoglobin solutions are more than mere blood substitutes. Promising effects on oxygen transport and the microcirculation need to be confirmed, and the results of continuing research are eagerly awaited.

Roundtable conference on tissue oxygenation in acute medicine, Brussels, Belgium, 14-16 March 1998.

* Nutritional tissue perfusion and tissue metabolic demand are heterogeneously distributed. * Oxygen diffusion occurs preferentially at the precapillary arteriolar level. * Determination of adequacy of tissue oxygenation requires local organ measurements. * While there remains considerable variability in individual RBC transfusion practices, a recent clinical trial questioned the efficacy of RBC transfusion to hemoglobin concentrations greater than 80 gm/l in patients without heart disease [63]. * RBC substitutes, including cell-free hemoglobin solutions and PFC solutions are efficacious, yet may exhibit a number of direct vascular effects.

The Hemodynamic Effects of Cell-Free Hemoglobin During General and Epidural Anesthesia

Although hemoglobin-based oxygen carriers (HBOC) are now being investigated, the effects of HBOC solutions during regional anesthesia have never been analyzed. Therefore, we investigated the hemodynamic changes after HBOC infusion during general anesthesia and thoracic epidural anesthesia. Sheep were assigned to three different groups: a) a control group with six unanesthetized sheep; b) six sheep with a halothane anesthesia (2.0 vol. % in oxygen); and c) six awake sheep with a thoracic epidural anesthesia with bupivacaine. After a period of stabilization, all 18 animals received 100 mg/kg of the HBOC pyridoxalated hemoglobin polyoxyethylene conjugate. The infusion of the HBOC caused a significant increase in mean arterial pressure and pulmonary artery pressure in both the control and epidural anesthesia groups. Anesthesia with halothane reduced the effects of the HBOC-solution on mean arterial pressure but did not abolish the increase in pulmonary artery pressure. Our results demonstrate that vasoconstriction caused by HBOC solutions is not abolished by epidural anesthesia, but halothane anesthesia may alter the hemodynamic effects of HBOC solutions. Implications We evaluated the effects of epidural anesthesia and halothane anesthesia on the vasoconstrictive properties of a cell-free hemoglobin solution. The vasoconstriction caused by a cell-free hemoglobin solution was similar in unanesthetized sheep and sheep with thoracic epidural anesthesia and was reduced in sheep with halothane anesthesia.

Glutaraldehyde Modification of Recombinant Human Hemoglobin Alters Its Hemodynamic Properties

Many cell-free hemoglobin solutions designed as oxygen-carrying therapeutics produce a hypertensive effect in animals. The response is likely due to oxidation of nitric oxide by hemoglobin. Since the site of oxidation may lie outside the vascular compartment, we tested the hypothesis that polymerization of hemoglobin, rHb1.1, by glutaraldehyde would attenuate the hypertensive response. Two products of the cross-linking reaction were isolated, a glutaraldehyde-derivatized monomer (mono-glxrHb) and a glutaraldehyde cross-linked polymer (poly-glxrHb), and evaluated for their effects on systemic hemodynamics in conscious rats. Administration of rHb1.1 caused a mean arterial pressure elevation of approximately 20 mm Hg and an increase in total peripheral resistance of approximately 30%. Administration of mono-glxrHb induced changes in mean arterial pressure and vascular resistance that were significantly diminished relative to those observed with rHb1.1. Poly-glxrHb elicited a mean arterial pressure response that was further reduced compared with that obtained with mono-glxrHb and a change in vascular resistance that was the same as the response to mono-glxrHb. These results suggest that rHb peripheral vasoconstriction elicited by rHb1.1 is significantly attenuated by glutaraldehyde modification of the hemoglobin monomer and that the effect of glutaraldehyde polymerization is likely due to surface modification and/or intramolecular cross-linking, rather than an increase in molecular size.

Continuous infusion of pyridoxalated hemoglobin polyoxyethylene conjugate in hyperdynamic septic sheep.

Cell-free hemoglobin solutions can scavenge nitric oxide and therefore increase mean arterial pressure (MAP). The present study investigated the effects of a continuous low-dose infusion of modified hemoglobin during ovine hyperdynamic sepsis. 13 sheep received a continuous infusion of live Pseudomonas aeruginosa bacteria for 48 h. Animals that survived the first 24 h of sepsis (n=12) were randomly assigned either to a treatment group that received 20 mg x kg(-1) x h(-1) pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) for 20 h or to a control group that received the same volume of the vehicle for 20 h. MAP increased in the treatment group during 4 h of PHP infusion 12.7+/-1.7 mmHg (p < .05) and after 20 h of PHP infusion MAP was still 12.4+/-2.1 mmHg above the MAP before starting PHP (p < .05). MAP in the control group did not change significantly from 24 h to 48 h of sepsis. No differences in regional blood flow were seen between groups. Bacterial counts in the spleen and kidney were lower in the treatment group than in the control group. Continuous low-dose infusion of PHP can normalize systemic vascular resistance and MAP for long periods without deterioration of regional blood flow or bacterial clearance.

Arterial Blood Pressure Responses to Cell-free Hemoglobin Solutions and the Reaction with Nitric Oxide

Changes in mean arterial pressure were monitored in rats following 50% isovolemic exchange transfusion with solutions of chemically modified hemoglobins. Blood pressure responses fall into three categories: 1) an immediate and sustained increase, 2) an immediate yet transient increase, or 3) no significant change either during or subsequent to exchange transfusion. The reactivities of these hemoglobins with nitric monoxide (.NO) were measured to test the hypothesis that different blood pressure responses to these solutions result from differences in .NO scavenging reactions. All hemoglobins studied exhibited a value of 30 microM-1 s-1 for both .NO bimolecular association rate constants and the rate constants for .NO-induced oxidation in vitro. Only the .NO dissociation rate constants and, thus, the equilibrium dissociation constants varied. Values of equilibrium dissociation constants ranged from 2 to 14 pM and varied inversely with vasopressor response. Hemoglobin solutions that exhibited either transient or no significant increase in blood pressure showed tighter .NO binding affinities than hemoglobin solutions that exhibited sustained increases. These results suggest that blood pressure increases observed upon exchange transfusion with cell-free hemoglobin solutions can not be the result of .NO scavenging reactions at the heme, but rather must be due to alternative physiologic mechanisms.

Cell-Free Hemoglobin as an Oxygen Carrier Removes Nitric Oxide, Resulting in Defective Thromboregulation

Development of a clinically safe and effective substitute for erythrocytes that is capable of efficient oxygen delivery in vivo has progressed to the stage where cross-linked hemoglobin preparations are now undergoing clinical trials.1 Such preparations withstand storage for prolonged periods of time, can be administered without the need for cross-matching, and are free of contamination by infectious agents. Utilization of cell-free hemoglobin as an erythrocyte substitute was initially hampered by nephrotoxicity and an affinity for oxygen that prevented efficient oxygen delivery to tissues.2 These disadvantages were overcome when Bunn and Jandl3 chemically cross-linked the hemoglobin molecule to produce stable hemoglobin oligomers that do not pass through the glomerular filtrate. In addition, Benesch and Benesch4 developed reagents that modified the 2,3-diphosphoglycerate binding site of hemoglobin, thereby reducing its oxygen affinity. Administration of cell-free hemoglobin solutions results in systemic vasoconstriction in research animals.5 This is thought to be a consequence of the high avidity of hemoglobin for nitric oxide (NO, endothelium-derived relaxing factor [EDRF]), which it binds and inactivates. The NO-hemoglobin interaction results in rapid formation of nitrite/nitrate and methemoglobin. This blocks vasodilation induced by …

Encapsulated Hemoglobin: Current Issues and Future Goals

The promise of encapsulation systems for the sequestration of hemoglobin has been the long-held belief that encapsulation more closely mimics nature's strategy for circulating hemoglobin, and could alleviate hemoglobin based toxicities and increase circulation persistence. Various polymers have been proposed to deliver hemoglobin. One approach toward the encapsulation of hemoglobin has been to employ biodegradable, biocompatible vehicles such as phospholipid vesicles, or liposomes. The majority of encapsulation work with hemoglobin over recent years has focused on liposome encapsulated hemoglobin with demonstrations of efficacy and safety in total and partial isovolemic and hypovolemic exchange models, hemodynamics, circulation persistence and organ biodistribution, processing methods, long term storage through freeze-drying, and serum changes and histopathological consequences following administration in small animals. The data collected thus far indicate that encapsulation of hemoglobin does significantly alter many of the traditionally observed effects following the administration of cell free hemoglobin solutions. Liposome encapsulated hemoglobin circulates for 20-24 hours in small animals and principally distributes to the liver and spleen. The significant accumulation of liposome encapsulated hemoglobin in these organs poses new questions for short and long-term effects on the reticuloendothelial system and macrophage function which are currently being addressed. In addition, transient hemodynamic and serum changes have been observed following the administration of liposome encapsulated hemoglobin. Many of these are similar to the effects observed following administration of liposomes without intravesicular hemoglobin and are dictated by liposome parameters such as surface charge and character, size, and lipid composition. Finally, fundamental large scale production issues such as encapsulation efficiency and particle size distribution must be optimized to facilitate the commercial development of encapsulated hemoglobin. These issues are discussed in the historical context of encapsulated hemoglobin and basic liposome research, current research status, and future challenges for the development of encapsulated hemoglobin as an artificial oxygen carrying fluid.