Polymerized Bovine Hemoglobin Research Articles

(43) - A Newly Produced Polymerized Bovine Hemoglobin and Hemoglobin-Based Heart Preservation Solution Prolonged Preservation Duration of Donor Heart in Cold Static Storage and Reduced Myocardial Injury After Allograft Transplantation

(43) - A Newly Produced Polymerized Bovine Hemoglobin and Hemoglobin-Based Heart Preservation Solution Prolonged Preservation Duration of Donor Heart in Cold Static Storage and Reduced Myocardial Injury After Allograft Transplantation

Use of Hemoglobin Based Oxygen Carrier HBOC-021 (Hemopure) As a Bridge during Emergencies in Patients Unable to Receive Blood Products: Experience at a Tertiary Care Center

IntroductionThere are about 1.1 million Jehovah's witnesses (JW) in the United States who do not accept blood transfusions due to religious beliefs. When these patients present with severe anemia, management is challenging and mortality high. Hemoglobin based Oxygen Carrier HBOC-201 has been available for compassionate use, but its use is limited due to lack of experience. Here we present the experience of a tertiary care center with HBOC-201 to better manage critically ill JW patients with anemia.Case159 y.o. JW female was admitted with diabetic ketoacidosis, right lower extremity gangrene, and sepsis. Labs showed WBC 17.4, Hgb 7.9, and platelets 434. Within hours, the patient went into septic shock requiring vasopressors and mechanical ventilation, AKI requiring dialysis, and transaminitis. Her Hgb dropped to 6.9. She was started on insulin drip and broad-spectrum antibiotics. Her clinical condition improved, vasopressors were discontinued and she was extubated. She subsequently had a below knee amputation. On post-operative day 2, her condition worsened. She developed fevers and vasopressors were restarted. Her Hgb dropped to 5.8. Erythropoietin injections and Iron supplementation were initiated. At this point, the surgical team planned for an above knee amputation for better control of the infection, with a pre-operative goal Hgb of 8. On post-op day 7, the patient's clinical status deteriorated, and she was re-intubated and found to have an embolic stroke. Her Hgb continued to drop to 5.3 and she subsequently received 2 units of HBOC in preparation for urgent surgical intervention for infection control. Her Hgb improved to 6.5 following administration of 2 units of HBOC. Despite the improvement in Hgb, she acutely worsened with increased vasopressor and ventilation requirement, severe acidosis, methemoglobinemia, elevated troponin (0.5), and hypoglycemia. On the next day, she went into cardiac arrest and died.Case249 y.o. JW male was admitted with acute pancreatitis. Labs showed WBC 12.9, Hgb 18.6, transaminitis and lipase >600. The patient's clinical status improved with conservative management but on day 3 developed hematemesis with a drop in Hgb to 9.7. He became lethargic and was subsequently intubated. Esophagogastroduodenoscopy (EGD) showed a gastroesophageal junction clot. Gastrointestinal bleeding persisted over the next 3 days with drop in Hgb to 5. EGD showed an ulcer with visible vessel. Apart from proton pump inhibitor drip, he was also started on Erythropoeitin , B12 and Iron Despite these measures, his Hgb continued to drop with a nadir of 3.3 and worsening clinical status requiring multiple vasopressors. On Day 9, HBOC was approved for compassionate use. The patient was infused 4 units of HBOC. Hgb improved to 5.2, and the patient was weaned off vasopressors. A repeat EGD with injection and cauterization of the bleeding vessel were performed. The patient received one unit of HBOC daily to keep Hgb>5 (figure 1) and the gastroduodenal artery was embolized. The patient developed complications from HBOC, including hypernatremia, alkalosis and methemoglobinemia successfully treated with ascorbic acid and methylene blue. Troponin was normal and EKG was normal. Two days later, he developed lactic acidosis, ventilator associated pneumonia, acute respiratory distress syndrome, shock and multiorgan failure and succumbed to his illness. During this time, his Hgb was stable at 6.1 and there was no further bleeding.ConclusionHBOC-021 (Hemopureâ) is a polymerized bovine hemoglobin. It has been used for management of anemia in JW patients under compassionate-use FDA guidelines with varying success. Our experience with HBOC shows that it can be used successfully in emergencies to hemodynamically stabilize patients. Our first patient showed an improvement in Hgb while our second patient attained a stable Hgb and recovered from his hemorrhagic shock. With clinical improvement, he could get an EGD and embolization done which controlled his bleeding. The side effects of HBOC are easily managed with supportive care and methylene blue. We believe that Intensivists and Hematologists should be aggressively involved in JW patients who urgently need but refuse blood transfusion. An institution based protocol should be available to rapidly obtain HBOC. Efforts should be made to minimize blood loss and the use of erythropoiesis stimulating agents should be maximized. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Balance between oxygen transport and blood rheology during resuscitation from hemorrhagic shock with polymerized bovine hemoglobin.

Alternatives to blood for use in transfusion medicine have been investigated for decades. An ideal alternative should improve oxygen (O2)-carrying capacity and O2 delivery and support microvascular blood flow. Previous studies have shown that large-molecular diameter hemoglobin (Hb)-based oxygen carriers (HBOCs) based on polymerized bovine Hb (PolybHb) reduce the toxicity and vasoconstriction of first-generation HBOCs by increasing blood and plasma viscosity and preserving microvascular perfusion. The objective of this study was to examine the impact of PolybHb concentration and therefore O2-carrying capacity and solution viscosity on resuscitation from hemorrhagic shock in rats. PolybHb was diafiltered on a 500-kDa tangential flow filtration (TFF) module to remove low-molecular weight (MW) PolybHb molecules from the final product. Rats were hemorrhaged and maintained in hypovolemic shock for 30 min before transfusion of PolybHb at 10 g/dL (PHB10), 5 g/dL (PHB5), or 2.5 g/dL (PHB2.5) concentration, to restore blood pressure to 90% of the animal's baseline blood pressure. Resuscitation restored blood pressure and cardiac function in a PolybHb concentration-dependent manner. Parameters indicative of the heart's metabolic activity indicated that the two higher PolybHb concentrations better restored coronary O2 delivery compared with the low concentration evaluated. Markers of organ damage and inflammation were highest for PHB10, whereas PHB5 and PHB2.5 showed similar expression of these markers. These studies indicate that a concentration of ~5 g/dL of PolybHb may be near the optimal concentration to restore cardiac function, preserve organ function, and mitigate the toxicity of PolybHb during resuscitation from hemorrhagic shock.NEW & NOTEWORTHY Large-molecular diameter polymerized bovine hemoglobin avoided vasoconstriction and impairment of cardiac function during resuscitation from hemorrhagic shock that was seen with previous hemoglobin-based O2 carriers by increasing blood viscosity in a concentration-dependent manner. Supplementation of O2-carrying capacity played a smaller role in maintaining cardiac function than increased blood and plasma viscosity.

Comprehensive characterization of tense and relaxed quaternary state glutaraldehyde polymerized bovine hemoglobin as a function of cross‐link density

Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this study, we prepared PolybHb in the T and relaxed (R) quaternary state with ultrahigh MW (>500 kDa) with varying cross-link densities, and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, auto-oxidation, and haptoglobin [Hp]-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e., cross-linker:bovine hemoglobin (bHb) molar ratio, gas-liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T- and R-state PolybHbs. Our results showed that, the duration of the fast-step auto-oxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHbs exhibited significantly higher bimolecular rate constants for binding to Hp and unimolecular O2 offloading rate constants compared to R-state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.

Ex-Vivo Normothermic Limb Perfusion With a Hemoglobin-Based Oxygen Carrier Perfusate.

Ex-vivo normothermic limb perfusion (EVNLP) has been proven to preserve limb viability better than standard cold storage. Perfusates containing packed red blood cells (pRBC) improve outcomes when compared to acellular perfusates. Limitations of pRBC-based perfusion include limited availability, need for cross match, mechanical hemolysis, and activation of pro-inflammatory proteins. Hemoglobin-based oxygen carrier (HBOC)-201 (Hemopure) is a solution of polymerized bovine hemoglobin, characterized by low immunogenicity, no risk of hemolytic reaction, and enhanced convective and diffusive oxygen delivery. This is a preliminary study on the feasibility of EVNLP using HBOC-201 as an oxygen carrier. Three porcine forelimb perfusions were performed using an established EVNLP model and an HBOC-201-based perfusate. The perfusion circuit included a roller pump, oxygenator, heat exchanger, and reservoir. Electrolytes, limb temperature, weight, compartment pressure, nerve conduction, and perfusion indicated by indocyanine green angiography and infra-red thermography were monitored. Histological evaluation was performed with hematoxylin and eosin and electron microscopy. Three limbs were perfused for 21.3±2.1hours. Muscle contractility was preserved for 10.6±2.4hours. Better preservation of the mitochondrial ultrastructure was evident at 12hours in contrast to crystallization and destruction features in the cold-storage controls. An HBOC-201-EVNLP produced outcomes similar to RBC-EVNLP with preservation of muscle contractility and mitochondrial structure.

Cardiac function during resuscitation from hemorrhagic shock with polymerized bovine hemoglobin-based oxygen therapeutic

Hemorrhage impairs myocardial contractile function and decreases oxygen delivery. This study investigates how polymerized bovine hemoglobin (PolyHb) solutions affect cardiac function after resuscitation from hemorrhagic shock (HS). Hamsters were hemorrhaged and resuscitated with PolyHb at 8.5 g/dL and 11.5 g/dL. Left ventricle (LV) function was assessed during shock and resuscitation using a miniaturize conductance catheter. PolyHb resuscitation had no beneficial effects in cardiac function; it increased cardiac afterload and systemic vascular resistance (SVR) of 46 and 116% for 8.5 and 11.5 g/dL, respectively. Study findings indicate that preclinical evaluation of cardiac function is essential to develop safe and efficacious alternatives to blood transfusion.

Transcriptional Suppression of Renal Antioxidant Enzyme Systems in Guinea Pigs Exposed to Polymerized Cell-Free Hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and plasma volume-expanding therapeutics though their potential to promote oxidative tissue injury has raised safety concerns. Using a guinea pig exchange transfusion model, we examined the effects of polymerized bovine hemoglobin (HbG) on the transcriptional regulation, activity, and expression of the renal antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). HbG infusion downregulated the mRNA levels for genes encoding SOD isoforms 1-3, GPx1, GPx3, GPx4, and CAT. This transcriptional suppression correlated with decreased enzymatic activities for SOD, CAT, and GPx. Immunostaining revealed decreased protein expression of SOD1, CAT, and GPx1 primarily in renal cortical tubules. DNA methylation analyses identified CpG hypermethylation in the gene promoters for SOD1-3, GPx1, GPx3, and GPx4, suggesting an epigenetic-based mechanism underlying the observed gene repression. HbG also induced oxidative stress as evidenced by increased renal lipid peroxidation end-products and 4-HNE immunostaining, which could be the result of the depleted antioxidant defenses and/or serve as a trigger for increased DNA methylation. Together, these findings provide evidence that the renal exposure to HbG suppresses the function of major antioxidant defense systems which may have relevant implications for understanding the safety of hemoglobin-based products.

Cotransplantation of Polymerized Hemoglobin Reduces β-Cell Hypoxia and Improves β-Cell Function in Intramuscular Islet Grafts.

Muscle is a promising alternative site for islet transplantation that facilitates rapid restoration of islet vascularization. However, the development of fibrosis suggests massive cellular death after transplantation. This study tested the hypothesis that islet graft function is limited by hypoxia-related death early after intramuscular transplantation, but that this can be overcome by cotransplantation of an oxygen carrier, that is, polymerized bovine hemoglobin (PolyHb). Two hundred islets were transplanted with or without different doses of PolyHb intramuscularly to nondiabetic C57BL/6 and diabetic C57BL/6 nu/nu mice. β-cell hypoxia and apoptosis were evaluated by immunohistochemistry after injection of the biochemical marker pimonidazole or by staining for caspase-3, respectively. Blood glucose concentrations were monitored for 30 days after islet transplantation and animals were then subjected to an intravenous glucose tolerance test. Substantial hypoxia was observed in control islet grafts during the first 4 days after transplantation. Cotransplantation of PolyHb resulted in a dose-dependent reduction of β-cell hypoxia, but β-cell apoptosis was only reduced by cotransplantation of low-dose PolyHb (0.03 mg/g body weight) due to the inflammatory effects of higher PolyHb concentrations. Cotransplantation of low-dose PolyHb resulted in improved islet graft function 30 days after transplantation in diabetic mice, with a glucose tolerance comparable to transplantation of 50% more islets. We conclude that cotransplantation of islets with PolyHb can be used to effectively bridge the critical hypoxic phase immediately after transplantation, improve islet graft function, and reduce the number of islets needed for successful intramuscular transplantation.

Down Selection of Polymerized Bovine Hemoglobins for Use as Oxygen Releasing Therapeutics in a Guinea Pig Model

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being developed as resuscitative fluids for use in multiple medical applications and in lieu of blood transfusion. However, cardiovascular, central nervous system, and renal adverse events have largely impeded progress. This has prompted a need to evaluate novel down selection approaches for HBOCs prior to in-depth preclinical and clinical safety testing. In the present study, polymerized bovine Hbs (PolybHbs) were prepared with increasing ratios of glutaraldehyde to bovine Hb (10:1, 20:1, 30:1, and 40:1). The optimal PolybHb candidate selection was based on a priori determined in vivo response to include a long circulating PolybHb with no measurable renal exposure, minimal cardiovascular response, limited oxidation to metHb in vitro, or in circulation and absence of acute end organ toxicity. Guinea pigs were dosed via a 50% blood for PolybHb exchange transfusion. Data suggested that the 30:1 preparation exhibited maximum circulatory exposure (AUC(0)(-∞)) with the lowest level of oxidation (plasma metHb formation) and minimal (< 10%) blood pressure elevation. Additionally, the 30:1 preparation was absent renal iron deposition as well as abnormal glomerular/tubular histopathology or serum creatinine elevation. Clearance pathways predominantly followed those consistent with endogenous Hb clearance based pathways. Therefore, data confirmed the ability to select a single PolybHb from a small library of HBOCs based on a priori determined characteristics. Moreover, the approach to down selection described could be applied to enhance the early predictability of human safety for this class of biological therapeutics to optimize for specific indications.

Pre-clinical studies using OxyVita hemoglobin, a zero-linked polymeric hemoglobin: a review

Hemoglobin-based oxygen carriers (HBOC) are being developed to provide the oxygen necessary in clinical situations when whole blood is not available. The safety and effectiveness of each HBOC must be determined before clinical approval. In the past several years animal studies have been conducted with zero-linked polymers to evaluate their effectiveness at delivering oxygen in vivo. Studies have addressed issues associated with interstitial extravasation, cerebral ischemia and blood flow, resuscitation, and coagulation interactions. Several of the investigations reviewed are based on early preparations of zero-linked polymerized bovine hemoglobins (ZL-HbBv), which contained a wide range of high-molecular-weight polymers. Recent studies using the Oxyvita product OxyVita Hb, which contains a more homogenous population (97%) of large-molecular-weight species (~17MDa), are also included in this review.

Synthesis, biophysical properties and pharmacokinetics of ultrahigh molecular weight tense and relaxed state polymerized bovine hemoglobins

Hemoglobin-based oxygen carriers (HBOC) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, late stage clinical results of polymerized hemoglobin (PolyHb) solutions hamper development. We synthesized two types of PolyHbs with ultrahigh molecular weights: tense (T) state PolyHb ( M W = 16.59 MDa and P 50 = 41 mm Hg) and relaxed (R) state PolyHb ( M W = 26.33 MDa and P 50 = 0.66 mm Hg). By maintaining Hb in either the T- or R-state during the polymerization reaction, we were able to synthesize ultrahigh molecular weight PolyHbs in distinct quaternary states with no tetrameric Hb, high viscosity, low colloid osmotic pressure and the ability to maintain O 2 dissociation, CO association and NO dioxygenation reactions. The PolyHbs elicited some in vitro RBC aggregation that was less than 6% dextran (500 kDa) but more than 5% human serum albumin. In vitro, T-state PolybHb autoxidized faster than R-state PolybHb as expected from previously reported studies, conversely, when administered to guinea pigs as a 20% exchange transfusion, R-state PolybHb oxidized faster and to a greater extent than T-state PolybHb, suggesting a more complex oxidative processes in vivo. Our findings also demonstrate that T-state PolybHb exhibited a longer circulating half-life, slower clearance and longer systemic exposure time compared to R-state PolybHb.

Differential Induction of Renal Heme Oxygenase and Ferritin in Ascorbate and Nonascorbate Producing Species Transfused with Modified Cell-Free Hemoglobin

Abstract Heme catabolism and iron sequestration systems play an important role in regulating the response to extracellular hemoglobin (Hb). We previously reported that extracellular Hb oxidizes more readily in the circulation of guinea pigs, a nonascorbate (AA)-producing species with similar plasma and tissue antioxidant status to humans, compared to rats, an AA-producing species. To determine whether these two species exhibit differences in heme catabolism and iron sequestration at the level of the kidney, we examined heme oxygenase (HO), H- and L-ferritin expression, nonheme iron deposition, and renal AA content following transfusion with polymerized bovine hemoglobin (HbG). Both species showed similar rates of hemoglobinuria but urinary HbG was significantly more oxidized in guinea pigs. HbG enhanced HO activity in both species but appeared greater and more sustained in guinea pigs. Conversely, rats showed a greater and more rapid induction of H- and L-ferritin as well as greater iron accumulation and AA content. Furthermore, ferrous and ferric iron deposits were detected in rats while only ferric iron was observed in guinea pigs. These findings suggest significant differences in the renal handling of HbG which may be important for understanding how endogenous antioxidant defenses may modulate the renal response to extracellular Hb.

Tissue oxygenation after exchange transfusion with ultrahigh-molecular-weight tense- and relaxed-state polymerized bovine hemoglobins

Hemoglobin (Hb)-based O(2) carriers (HBOCs) constitute a class of therapeutic agents designed to correct the O(2) deficit under conditions of anemia and traumatic blood loss. The O(2) transport capacity of ultrahigh-molecular-weight bovine Hb polymers (PolybHb), polymerized in the tense (T) state and relaxed (R) state, were investigated in the hamster chamber window model using microvascular measurements to determine O(2) delivery during extreme anemia. The anemic state was induced by hemodilution with a plasma expander (70-kDa dextran). After an initial moderate hemodilution to 18% hematocrit, animals were randomly assigned to exchange transfusion groups based on the type of PolybHb solution used (namely, T-state PolybHb and R-state PolybHb groups). Measurements of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O(2) levels were performed at 11% hematocrit. Both PolybHbs were infused at 10 g/dl, and their viscosities were higher than nondiluted blood. Restitution of the O(2) carrying capacity with T-state PolybHb exhibited lower arterial pressure and higher functional capillary density compared with R-state PolybHb. Central arterial O(2) tensions increased significantly for R-state PolybHb compared with T-state PolybHb; conversely, microvascular O(2) tensions were higher for T-state PolybHb compared with R-state PolybHb. The increased tissue Po(2) attained with T-state PolybHb results from the larger amount of O(2) released from the PolybHb and maintenance of macrovascular and microvascular hemodynamics compared with R-state PolybHb. These results suggest that the extreme high O(2) affinity of R-state PolybHb prevented O(2) bound to PolybHb from been used by the tissues. The results presented here show that T-state PolybHb, a high-viscosity O(2) carrier, is a quintessential example of an appropriately engineered O(2) carrying solution, which preserves vascular mechanical stimuli (shear stress) lost during anemic conditions and reinstates oxygenation, without the hypertensive or vasoconstriction responses observed in previous generations of HBOCs.

Differential sensitivities of pulmonary and coronary arteries to hemoglobin-based oxygen carriers and nitrovasodilators: Study in a bovine ex vivo model of vascular strips

Vasoconstriction is a major adverse effect of first and second generation hemoglobin-based oxygen carriers (HBOCs) that hinders their development as blood substitute. However, intravenous infusion of HBOC-201 (second generation) to patients induces significant pulmonary hypertension without significant coronary vasoconstriction. We compared contractile responses of isolated bovine pulmonary and coronary arterial strips to HBOC-201 and HBOC-205LL.LT.MW600 (third generation), polymerized bovine hemoglobins of different molecular weight, and their attenuation by nitroglycerin, sodium nitroprusside (SNP), and sodium nitrite. Pulmonary arteries developed negligible basal tone, but exhibited HBOC-dependent amplification of phenylephrine-induced contractions. In contrast, coronary arteries developed significant basal tone, and exhibited HBOC-dependent constant force increment to serotonin-induced contractions. Therefore, relative to basal tone, HBOC-induced contractions were greater in pulmonary than coronary arteries. Furthermore, HBOC-205LL.LT.MW600 appeared to be less vasoactive than HBOC-201. Unexpectedly, pulmonary and coronary arteries exhibited differential sensitivities to nitrovasodilators in parallel with their differential sensitivities to HBOC. However, SNP and sodium nitrite induced significant methemoglobin formation from HBOC, whereas nitroglycerin did not. These results suggest that phenotypic differences between pulmonary and coronary vascular smooth muscle cells could explain the differential hypertensive effects of HBOC on pulmonary and coronary circulation in patients. Among the three nitrovasodilators investigated, nitroglycerin appears to be the most promising candidate for attenuating HBOC-induced pulmonary hypertension in older HBOCs.

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.

POLYMERIZED BOVINE HEMOGLOBIN CAN IMPROVE SMALL-VOLUME RESUSCITATION FROM HEMORRHAGIC SHOCK IN HAMSTERS

Systemic and microvascular hemodynamic responses to hemorrhagic shock volume resuscitation with hypertonic saline followed by infusion of polymerized bovine hemoglobin (PBH) at different concentrations were studied in the hamster window chamber model to determine the role of plasma oxygen-carrying capacity and vasoactivity during resuscitation. Moderate hemorrhagic shock was induced by arterial controlled bleeding of 50% of blood volume (BV), and a hypovolemic state was maintained for 1 h. Volume was restituted by infusion of hypertonic saline (7.5% NaCl), 3.5% of BV, followed by 10% of BV of PBH at 2 different concentrations. Resuscitation was followed for 90 min and was carried out using 13 gPBH/dL (PBH13), PBH diluted to 4 gPBH/dL in albumin solution at matching colloidal osmotic pressure (PBH4), and an albumin-only solution at matching colloidal osmotic pressure (PBH0). Systemic parameters, microvascular hemodynamics, and functional capillary density were determined during hemorrhage, hypovolemic shock, and resuscitation. The PBH13 caused higher arterial pressure without reverting vasoconstriction and hypoperfusion. The PBH4 and PBH0 had lower MAP and partially reverted vasoconstriction. Only treatment with PBH4 restored perfusion and functional capillary density when compared with PBH13 and PBH0. Blood gas parameters and acid-base balance recovered proportionally to microvascular perfusion. Tissue PO2 was significantly improved in the PBH4 group, showing that limited restoration of oxygen-carrying capacity is beneficial and compensates for the effects of vasoactivity, a characteristic of molecular hemoglobin solutions proposed as blood substitutes.

Prevention of the Pulmonary Vasoconstrictor Effects of HBOC-201 in Awake Lambs by Continuously Breathing Nitric Oxide

Hemoglobin-based oxygen-carrying solutions (HBOC) provide emergency alternatives to blood transfusion to carry oxygen to tissues without the risks of disease transmission or transfusion reaction. Two primary concerns hampering the clinical acceptance of acellular HBOC are the occurrence of systemic and pulmonary vasoconstriction and the maintenance of the heme-iron in the reduced state (Fe2+). We recently demonstrated that pretreatment with inhaled nitric oxide prevents the systemic hypertension induced by HBOC-201 (polymerized bovine hemoglobin) infusion in awake mice and sheep without causing methemoglobinemia. However, the impact of HBOC-201 infusion with or without inhaled nitric oxide on pulmonary vascular tone has not yet been examined. The pulmonary and systemic hemodynamic effects of breathing nitric oxide both before and after the administration of HBOC-201 were determined in healthy, awake lambs. Intravenous administration of HBOC-201 (12 ml/kg) induced prolonged systemic and pulmonary vasoconstriction. Pretreatment with inhaled nitric oxide (80 parts per million [ppm] for 1 h) prevented the HBOC-201--induced increase in mean arterial pressure but not the increase of pulmonary arterial pressure, systemic vascular resistance, or pulmonary vascular resistance. Pretreatment with inhaled nitric oxide (80 ppm for 1 h) followed by breathing a lower concentration of nitric oxide (5 ppm) during and after HBOC-201 infusion prevented systemic and pulmonary vasoconstriction without increasing methemoglobin levels. These findings demonstrate that pretreatment with inhaled nitric oxide followed by breathing a lower concentration of the gas during and after administration of HBOC-201 may enable administration of an acellular hemoglobin substitute without vasoconstriction while preserving its oxygen-carrying capacity.

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

A mathematical model was developed to study O(2) transport in a convection enhanced hepatic hollow fiber (HF) bioreactor, with hemoglobin-based O(2) carriers (HBOCs) present in the flowing cell culture media stream of the HF lumen. In this study, four HBOCs were evaluated: PEG-conjugated human hemoglobin (MP4), human hemoglobin (hHb), bovine hemoglobin (BvHb) and polymerized bovine hemoglobin (PolyBvHb). In addition, two types of convective flow in the HF extra capillary space (ECS) were considered in this study. Starling flow naturally occurs when both of the ECS ports are closed. If one of the ECS ports is open, forced convective flow through the ECS will occur due to the imposed pressure difference between the lumen and ECS. This type of flow is referred to as cross-flow in this work, since some of the fluid entering the HF lumen will pass across the HF membrane and exit via the open ECS port. In this work, we can predict the dissolved O(2) concentration profile as well as the O(2) transport flux in an individual HF of the bioreactor by solving the coupled momentum and mass transport equations. Our results show that supplementation of the cell culture media with HBOCs can dramatically enhance O(2) transport to the ECS (containing hepatocytes) and lead to the formation of an in vivo-like O(2) spectrum for the optimal culture of hepatocytes. However, both Starling flow and cross-flow have a very limited effect on O(2) transport in the ECS. Taken together, this work represents a novel predictive tool that can be used to design or analyze HF bioreactors that expose cultured cells to defined overall concentrations and gradients of O(2).

Decreased Damage From Transient Focal Cerebral Ischemia by Transfusion of Zero-Link Hemoglobin Polymers in Mouse

Transfusion of large polymers of hemoglobin avoids the peripheral extravasation and hypertension associated with crosslinked tetrameric hemoglobin transfusion and may be more effective in rescuing brain from focal ischemia. Effects of transfusion of high-oxygen-affinity hemoglobin polymers of different weight ranges were determined. Hypervolemic exchange transfusion was performed during 2 hours of middle cerebral artery occlusion in mice. Compared to transfusion with a 5% albumin solution or no transfusion, infarct volume was reduced 40% by transfusion of a 6% solution containing hemoglobin polymers in the nominal range 500 to 14 000 kDa. Infarct volume was not significantly reduced by transfusion of a lower concentration of 2% to 3% of this size range of polymers, 6% hemoglobin solutions without removal of polymers <500 kDa or >14000 kDa, or crosslinked hemoglobin tetramers with normal oxygen affinity. Exchange transfusion with the 6% solution of the 500 to 14 000 kDa hemoglobin polymers did not improve the distribution of cerebral blood flow during focal ischemia and, in mice without ischemia, did not affect flow to brain or other major organs. An intermediate size range of polymerized bovine hemoglobin possessing high oxygen affinity appears optimal for rescuing mouse brain from transient focal cerebral ischemia. A minimum concentration of a 6% solution is required, the rescue is superior to that obtained with crosslinked tetrameric hemoglobin possessing normal oxygen affinity, and tissue salvage is not associated with increased blood flow. This polymer solution avoids the adverse effects of severe renal and splanchnic vasoconstriction seen with crosslinked tetrameric hemoglobin.