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The goal of this study was to investigate whether hemoglobin- based oxygen carrier (HBOC) attenuated ischemia/reperfusion (I/R)-induced kidney injury. Male SD rats were randomly divided into a sham group, hR group, and HBOC group (injection of 0.1 gHb/kg PolyPHb). The ischemia was induced by bilateral renal pedicle cross-clamping for 45mm. Then the clamp was released to allow 24h reperfusion. Without increasing blood pressure, PolyPHb reduced the blood urea nitrogen and creatinine in plasma and attenuated the tumor necrosis factor-a and interleukin-8 in kidney tissue. Therefore, our findings suggest that PolyPHb could reduce kidney injury after I/R injury, and this effect was probably associated with the depressed inflammatory response. [ABSTRACT FROM AUTHOR]

To investigate whether polymerized human placenta hemoglobin (PolyPHb) improved hemodynamic parameter recovery and cardiac function after hemorrhagic shock, a mean arterial pressure (MAP) of 30 mmHg was maintained for 60min. Then, all the rats were randomly resuscitated with hetastarch (HES), shed blood (Whole Blood), or PolyPHb (PolyPHb). The MAP was greatly improved by PolyPHb, but it was still lower than that of the Whole Blood group. Meanwhile, the cardiac function and enzyme releases in the PolyPHb group were similar to the HES group. Therefore, our findings suggest that PolyPHb moderately improved hemodynamic recovery and provided little cardioprotective effect in hemorrhagic shock. [ABSTRACT FROM AUTHOR]

This study aims to examine the contribution of PEG-conjugated hemoglobin combined with cisplatin to the expression of HIF-1α and MDR1 in a tumor xenograft model. Cervical carcinoma models were assigned to 4 groups and treated respectively: group 1(control); group 2, cisplatin; group 3, PEG-Hb; group 4 cisplatin plus PEG-Hb. 4 weeks later, tumor volume and MVD was significantly decreased in group 4 compared with other groups. Lower expression of HIF-1α and MDR1 were detected in group4. Taken together, our data indicated that PEG-Hb plus cisplatin can promote tumor tissue oxygenation and enhance the chemotherapy sensitivity. HIF-1α regulated MDR1 pathway correlated with this process. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: To investigate the cardioprotective effect of polymerized human placenta hemoglobin (PolyPHb) for acute myocardial ischemia rat heart. Methods: Myocardial infarcts (MI) model was set up in SD rats by permanent ligation of the left anterior descending coronary artery (LDA). The rats were divided randomly into 3 groups with each group of 20 rats: (A) the control group with the administration of Ringer's lactated solution at a dose of 2.5ml/kg); (B) PolyPHb group,PolyPHb solution at a dose of 0.16g Hb/kg and (C), PolyPHb++CAT++SOD group, PolyPHb solution at a dose of 0.16g Hb/kg and catalase (CAT) solution and superoxide dismutase (SOD) solution at a dose of 1681 U/kg and 528000 U/kg, respectively. Each rat received treatments via caudal vein, once a day for 7 days. Qualitative evaluations were made based on the reading of cardiac troponin T (cTnT), the myocardial infarction size (MIS) derived from the staining of myocardium tissue, and the pathological changes in infarct area. The ischemia changes of cardiomyocytes were determined by haematoxylin - basic fuchsin - picric acid (HBFP) staining and the apoptosis of cardiomyocytes were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL). Results: Compared to the control group, PolyPHb greatly decreased the cTnT (p < 0.05), MIS (p < 0.05), and the size of myocardial ischemia (p < 0.05). PolyPHb ++ CAT ++ SOD decreased the △△ST change (P < 0.05), cTnT (P < 0.01), MIS (p < 0.05), the pathological scores (p < 0.01), the size of myocardial ischemia (p < 0.01), and the apoptosis level (p < 0.01). Conclusion: Our study demonstrated that adding PolyPHb improves cardiac functional recovery and reduces myocardial infarction of rat heart. [ABSTRACT FROM AUTHOR]

Abstract: This study was to investigate whether polymerized human placenta hemoglobin (PolyPHb) given before ischemia protects in vivo rat heart function against ischemia/reperfusion (I/R) injury. Forty-five male Sprague-Dawley rats were randomly divided (n == 15 per group) into a sham group, control group (pretreatment with Lactated Ringer's solution), or PolyPHb group (pretreatment with 0.1gHb/kg PolyPHb). Rat hearts were subjected to 30-min ischemia by occlusion of left anterior descending, followed by 2-hr reperfusion. As compared to the control group, PolyPHb preserved cardiac function and reduced cardiac troponin-I release and histopathological changes. Therefore, PolyPHb pretreatment provided a profound cardioprotective effect on the in vivo rat heart. [ABSTRACT FROM AUTHOR]

Abstract: Polymerized Porcine Hemoglobin (pPolyHb), a hemoglobin-based oxygen carrier (HBOC), was developed as a potential red blood substitute for clinical applications. Assessment of its effects on the immune system is an important component of the overall safety evaluation of HBOC. For this purpose, we assessed three inflammation indicators, including complement C3a, IL-6, and TNF-α in cultured cells and in a rat model when pPolyHb was incubated or administrated with the cells/animals. Our results suggested that the levels of these three indicators were not statistically changed upon pPolyHb stimulation, indicating that pPolyHb is not immunotoxic to cells and animals in this aspect. [ABSTRACT FROM AUTHOR]

Abstract: We have previously reported that derivatization of hemoglobin with periodate-modified sugar derivatives such as oxidized adenosine triphosphate (oATP) leads to an increase in prooxidant reactivity at the heme. Here, we report that copolymerization of hemoglobin with serum albumin alleviates this problem completely, to the extent where the copolymer even has a slightly lower autooxidation rate compared to native hemoglobin. A similar, although not as potent, effect is obtained when hemoglobin is derivatized with oATP in the presence of small-molecule antioxidants instead of albumin. [ABSTRACT FROM AUTHOR]

Experimental data, obtained during the course of several years, are connected into a coherent picture, which may help research for the development of HBOCs as therapeutic agents. Oxygen affinity, scavenging of nitric oxide, and yield of production of hemoglobin based oxygen carriers were the areas under consideration. [ABSTRACT FROM AUTHOR]

Abstract: The objective of the present study is to evaluate the pharmacodynamic properties of polymerized porcine hemoglobin (pPolyHb) in an exchange transfusion model. Each of two groups of rats received a volume of pPolyHb or hetastarch that equalled 120--140% of estimated total blood volume (70 ml/kg) exchange transfusion. The results showed pPolyHb retained hemodynamic stability and exhibited superior volume expansion capability. Furthermore, pPolyHb effectively reverse anaerobic metabolism caused by a large amount of volume exchange. In comparison with hetastarch, pPolyHb increased blood oxygen content and tissue oxygenation. All these properties contribute to a higher effectiveness in sustaining the lives of rats in pPolyHb group. [ABSTRACT FROM AUTHOR]

The propensity of site-specific carboxymethylation and Propylation of Val-1(αα) of Hb to attenuate the reductive hexaPEGylation-induced dissociation of tetramers has been investigated. Only reductive Propylation of Val-1(αα), which increases the stability of oxy Hb, attenuates the reductive hexaPEGylation-induced dissociation. Increasing the stability of the oxy conformation of Hb by chemical or genetic approaches is a strategy to generate PEGylated Hbs with native-like tetramer stability using direct PEGylation platforms. This new approach and EAF-PEGylation are the only two alternate PEGylation strategies available to design stable second-generation vasoinactive uncrosslinked PEGylated Hbs with native-like tetramer stability. [ABSTRACT FROM AUTHOR]

Human plasma fraction IV is an intermediate precipitate during the production of human serum albumin using cold ethanol method. Haptoglobin locates in this fraction can be purified for various applications. A new process integration of polyethylene glycol (PEG) precipitation and ion-exchange chromatography (IEC) was developed for purification of haptoglobin, which could effectively purify the haptoglobin from 16.6% to 95%. The recovery of the new process was 58.2% in comparison to 30.3% of the conventional affinity chromatography. Furthermore, 175 mg haptoglobin production in a scaled-up process showed the method to be simple, fast, and low-cost. [ABSTRACT FROM AUTHOR]

OxyVita-zero-link polymerized hemoglobin (OxyVita Hb) is a novel generation hemoglobin based oxygen carrier (HBOC). Our focus in this paper is to address the question of 'Why is OxyVita Hemoglobin different than the previous generation of HBOCs?' Several critical related topics will be discussed: 1) OxyVita's unique chemistry; 2) the introduction of a viable powder form of OxyVita hemoglobin for dissolution in IV water or other IV fluids; 3) the physiochemical characteristics of OxyVita hemoglobin preparations; 4) the ability to formulate different products based upon specific applications; and 5) the important storage properties essential for use in a wide range of geographical locations. [ABSTRACT FROM AUTHOR]

The article presents abstracts on medical-related topics which include the effect of cell-free hemoglobin on the cerebral circulation, the fusion of hemoglobin for blood substitute purposes, and the effects of sodium nitrite on vasoactivity associated with the hemoglobin based oxygen carrier HBOC-201 in controlled hemorrhage in swine.

Abstract: The effect of transfusion of PEGylated hemoglobin (PEG-Hb) was evaluated in anesthetized rats subjected to 2 hours of focal cerebral ischemia and 1 day of reperfusion. PEG-Hb was stored in the carboxy state (PEG-COHb) to reduce autooxidation and increase the shelf life. Transfusion of 10 ml/kg of PEG-COHb at 20 minutes of ischemia did not alter arterial blood pressure or increase red cell flux in the ischemic core. Plasma hemoglobin increased to only 0.6 g/dL, yet infarct volume was markedly decreased and neurological deficits were improved. We conclude that early topload transfusion of PEG-COHb protects the brain from ischemic stroke. [ABSTRACT FROM AUTHOR]

This study was designed to investigate whether polymerized human placenta hemoglobin (PolyPHb) pretreatment provided protection to the heart after ischemia/reperfusion (I/R) injury. After 10-min basal perfusion, isolated Sprague-Dawley rat hearts were pretreated with 0.1 gHb/dL PolyPHb and subjected to I/R injury. PolyPHb pretreatment greatly reduced the decreases in left ventricular developed pressure (LVDP), maximum LVDP increase and decrease rate (±dp/dt), and the increase in left ventricular end-diastolic pressure (LVEDP) as compared to the control group. Moreover, the myocardial infarction and troponin-I release were significantly reduced in the pre-HBOCs group. Therefore, PolyPHb pretreatment was protective to the isolated I/R heart. [ABSTRACT FROM AUTHOR]

A zero-linked polymeric hemoglobin (OxyVita Hb) has been developed for application as an acellular therapeutic hemoglobin-based-oxygen-carrier (HBOC). For effective and safe oxygen binding, transport and delivery, an HBOC must meet essential molecular requirements related to its structural integrity and redox stability. OxyVita™ is a super polymer possessing an average M.wt. of 17 × 106 Da. Structural integrity was determined by unfolding studies of OxyVita™ in the presence of increasing concentrations of urea. The unfolding midpoints (D1/2) of different preparations of OxyVita™ (solution and powder forms) were compared to Lumbricus Hb (LtHb) and Arenicola Hb (ArHb), natural acellular polymeric hemoglobins, which are serving as models for an effective and safe acellular HBOC. Reduction studies of OxyVita Hb using endogenous reducing agents were also investigated. Results from these studies indicate that: 1) OxyVita Hb exhibits greater resistance to conformational change than either LtHb or ArHb in the reduced (oxyHb) state; and 2) the reduction of met OxyVita Hb to oxyHb occurs slowly in the presence of either ascorbic acid (70% reduction in 560 min.) or β-NADH (40% reduction in 90 min.). These studies provide consistent evidence that OxyVita Hb possesses physiochemical properties that exhibit structural integrity and redox behavior necessary for functioning as an effective and safe HBOC within clinical applications. These results are in agreement with observations made by other investigators as to the reduction in heme-loss of OxyVita Hb, essential for the reversible binding/release of molecular oxygen within the circulatory system. [ABSTRACT FROM AUTHOR]

Liposome-encapsulated hemoglobin (LEH) based on a novel, synthetic, non-phospholipid was developed, and evaluated for cerebral energy metabolism in a 40% hemorrhage rat model. The markers of tissue energetics were monitored by 1H- and 31P-magnetic resonance spectroscopy (MRS). After hemorrhage, 1H-MRS showed an increase in the levels of lactate and pyruvate. These markers returned to baseline values following LEH resuscitation. Both LEH and saline were able to exert a neuron-protective effect as indicated by the recovery of N-acetylaspartate. 31P MRS showed a fall in phosphocreatine after hemorrhage, which upon LEH or saline resuscitation returned to the baseline values. Similarly, inorganic phosphate increased after bleeding, but returned to normal after resuscitation. LEH resuscitation also recovered β-ATP levels, but saline resuscitation provided only a modest recovery. The results indicate the utility of MRS to monitor cerebral metabolism in hemorrhage/resuscitation. The data is also supportive of the new LEH formulation as an oxygen carrier. [ABSTRACT FROM AUTHOR]

Crosslinking of ultrapure hemoglobin, crystalline catalase, and superoxide dismutase resulted in a soluble nanodimensional complex of polyhemoglobin-catalase-superoxide dismutase. A less expensive and more convenient way is to crosslink bovine stroma-free hemolysate (stroma-free hemolysate) that already contains hemoglobin, catalase, and superoxide dismutase into polyhemoglobin with catalase and superoxide dismutase activities (stroma-free polyhemolysate) [21]. The objective of the present study is to evaluate the immunological properties of this stroma-free polyhemolysate. Each of three groups of rats received weekly subcutaneous injections of one of the stroma-free polyhemolysate, stroma-free hemolysate, and saline for four weeks. One week after the four cycles of weekly immunization, serum and plasma were collected for C3a complement activation tests and Ouchterlony antibody-antigen precipitation tests, respectively. Results show that stroma-free polyhemolysate retained significant antioxidant enzyme activity. The C3a complement activation test and Ouchterlony test show that four weekly subcutaneous injections of bovine stroma-free polyhemolysate did not result in any immunological reaction in rats when tested this way. [ABSTRACT FROM AUTHOR]

In addition to heme-irons, reactive (β-globin thiols (βCys93s) of hemoglobin (Hb) also have been shown to interact with endogenous nitric oxide (NO) thereby contributing to vascular tone regulation. What relative roles do these NO binding sites contribute to the overall Hb-mediated vasoactivity? Several test Hbs with either or both the NO binding sites preliganded or blocked were prepared and tested in a rat thoracic aortic ring model. Hbs tested were: NEM-Hb (ferrous Hb with masked thiols), HbNO (ferrous Hb preliganded with NO), Hb+CN (ferric Hb liganded with CN−), NEM-HbNO and NEM-Hb+CN (Hbs with both heme-iron and cysteine sites preliganded or blocked). Typically, >0.2 μM control Hb significantly increased isometric tension in agonist stimulated vessel rings (58.1 ±7.0% over baseline). At comparable concentrations, NEM-Hb also caused a significant contraction (50.7±9.5%) while HbNO and Hb+CN did not (−5.5±6.0% and −3.7±4.6%, respectively). For these Hbs, masking thiols as well did not significantly alter respective vascular effects. Ferrous sperm whale myoglobin (Mb), which has no reactive thiol, elicited a significant contraction (55.1±13.2%) while metMb did not (−0.8±3.2%), suggesting the relative importance of heme-iron ligand and oxidation state in Hb vasoactivity. Additionally, ferrous or ferric equine heart cytochrome-C, a heme protein with no readily available heme-iron and cysteine binding sites, did not elicit notable contraction. Human Hb variants in which (βCys93s are deleted or substituted with non-cysteine residues did not reveal any documented significant hemodynamic abnormalities. These results indicate that reactive globin-thiols do not appear to play a prominent role relative to heme-irons in Hb-mediated vasoconstriction. [ABSTRACT FROM AUTHOR]

The Labscale tangential flow diafiltration system in conjunction with polyethersulfone Biomax filters (both purchased from Millipore Inc.) were used in the following studies: (1) A crucial step in the preparation of polyhemoglobin (polyHb) is the removal of tetrameric hemoglobin (Hb), which can cause adverse side-effects. The efficiency for this depends on the integrity of the 100kDa filters. Those with lower integrity were not effective whereas those with a higher integrity of 0.075ml/min were more effective. A filter with an integrity of 0.075ml/min can reduce the initial 11.2% tetrameric Hb concentration to 1.3% using a flow rate of 0.2ml/min. Higher flow rate of 2.2ml/min was not as effective. (2) Filtration with a 100kDa filter was quick and efficient in separating stroma-free hemoglobin solutions from cellular debris from hemolysate. (3) Both 5kDa and 100kDa filters are efficient in concentrating hemoglobin solutions. [ABSTRACT FROM AUTHOR]

A new procedure was developed to obtain high-quality polymerized human hemoglobin by modifying purified hemoglobin with PLP and polymerized with GDA. Comparing polymerized hemoglobin products obtained from different methods, the product from the new procedure has similar physical, chemical, and biological properties in the molecular distribution, methemoglobin concentration, oxygen carrier capacity, P50 and spectral analysis. Furthermore, the new procedure of modification after polymerization can save PLP greatly, and significantly reduce the cost. So the procedure of modification after polymerization is a better way in research and production. [ABSTRACT FROM AUTHOR]

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-β95 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. [ABSTRACT FROM AUTHOR]

Objective: To investigate the cardioprotective effect of polymerized human placenta hemoglobin (PolyPHb) to rat heart subjected to 8-hour hypothermic storage and 2-hour normothermic reperfusion. Methods and Results: Isolated rat hearts were perfused with Langendorff model; after 30 minutes of baseline, the hearts were arrested and stored by St. Thomas' solution (STS) without (STS group) or with 0.5 gHb/dL PolyPHb (PolyPHb group) at 4°C for 8 hours, then reperfused for 2 hours. Compared with STS group, PolyPHb in STS greatly improved the recovery of left ventricular developed pressure (LVDP), maximum LVDP increase and decrease rate (±dp/dt), coronary flow rate (CF). Also, both the cardiac enzyme release, including creatine kinase (CK) and lactate dehydrogenase (LDH), and myocardial infarction size were significantly reduced in PolyPHb group. Conclusion: Our study demonstrated that the PolyPHb was beneficial to improving cardiac functional recovery and reducing myocardial infarction of 8-hour hypothermic stored rat heart. [ABSTRACT FROM AUTHOR]

Establishing an acceptable safety and efficacy profile for hemoglobin based oxygen carriers, HBOCs, is essential. Understanding the data that describes an HBOC “safety profile” requires consideration beyond counting “events” typically associated with intent to treat analysis. The imputation of causation from numerical counts alone, without clinical context, is an incomplete description of a complex situation. Clinical contextualization provides a greater understanding of the clinico-pathological processes involved. Generating alternative hypotheses for the origin of the events, apart from the drug, patient co-morbidities, situational and disease demands, could include protocol design, failure to provide mitigation strategies, patient management issues, or inadequate education of investigators. How clinical contextualization can provide insight for the interpretation of significant safety profile events is discussed in the context of a large phase III clinical trial where an appreciation of factors underlying the differences between intent to treat analysis and other approaches is discussed. [ABSTRACT FROM AUTHOR]

Angiogenesis, an essential event involved in a tumor's progression and metastasis, is regulated by hypoxia. Hypoxia widely exists in solid tumors due to the abnormal vasculature of tumor tissue and insufficiency of tissue oxygenation. We speculate that hemoglobin-based oxygen carriers (HBOCs) can attenuate tissue hypoxia, thereby suppressingthe angiogenesis in solid tumor in the context that HBOCs have the ability to increase tissue oxygenation. In the present study, PEG-conjugated hemoglobin solution (0.3 g/kg i.v. or 0.6 g/kg i.v.) was intravenously administrated to BALB/c nude mice bearing the cervical tumor twice a week with or without the treatment of cisplatin (5mg/kg i.p.) to investigate whether PEG-conjugated hemoglobin has a chemo-sensitization effect though anti-angiogenesis pathway. Tumor volume was measured every three days and tumor hypoxia was detected by immunohistochemistry for Hypoxyprobe™-1. Anti-angiogenic effect was accessed by detection of mRNA and protein levels of vascular endothelial growth factor (VEGF), the most important angiogenic factor. Our results showed that high concentration of PEG-conjugated hemoglobin solution significantly impeded the growth of tumor when compared with the control group. Moreover, VEGF expression was declined when treated with PEG-conjugated hemoglobin, possibly through the HIF regulation system. Collectively, treatment of PEG-conjugated hemoglobin combination with cisplatin has an antiangiogeic effect, but the underlying mechanism should be further studied. [ABSTRACT FROM AUTHOR]

PEGylation induced increase in colloidal osmotic pressure (COP) of Hb, one of the unique molecular properties of PEG-Hb conjugates, facilitates the neutralization of the vasoconstrictive activity of acellular Hb is a function of chemistry of conjugation of PEG-chains. The dependence of COP with the chemistry is a consequence of PEGylation induced weakening of interdimeric interactions. The conservative Extension Arm Facilitated (EAF) PEGylation exerts least influence on lowering the tetramer stability as compared to direct PEGylation. We have now designed a new, non-conservative EAF PEGylation that uses acylation chemistry to introduce the extension arm onto proteins to delineate the role of the extension arm and of the charge at the site of attachment in PEG-Hb conjugate on tetramer stability. The non-conservative EAF PEGylation does not lower the tetramer stability just as the non-conservative direct PEGylation of Hb. The impact of the extension arm in PEGylated Hb in terms of their structure and potential significance of higher tetramer stability of PEG-Hb conjugates generated by EAF-PEGylation in the in vivo toxicity and in the design of oxygen carrying plasma expander has been discussed. [ABSTRACT FROM AUTHOR]

This study is to investigate the long-term effects of PEG-PLA nano artificial cells containing hemoglobin (NanoRBC) on renal function and renal histology after 1/3 blood volume top loading in rats. The experimental rats received one of the following infusions: NanoRBC in Ringer lactate, Ringer lactate, stroma-free hemoglobin (SFHB), polyhemoglobin (PolyHb), autologous rat whole blood (rat RBC). Blood samples were taken before infusions and on days 1, 7 and 21 after infusions for biochemistry analysis. Rats were sacrificed on day 21 after infusions and kidneys were excised for histology examination. Infusion of SFHB induced significant decrease in renal function damage evidenced by elevated serum urea, creatinine and uric acid throughout the 21 days. Kidney histology in SFHb infusion group revealed focal tubular necrosis and intraluminal cellular debris in the proximal tubules, whereas the glomeruli were not observed damaged. In all the other groups, NanoRBC, PolyHb, Ringer lactate and rat RBC, there were no abnormalities in renal biochemistry or histology. In conclusion, injection of NanoRBC did not have adverse effects on renal function nor renal histology. [ABSTRACT FROM AUTHOR]

A dynamic heterogeneous phase polymerization reaction is found to be efficient for controllable cross-link of hemoglobin with glutaraldehyde. The selective absorption of the immobile phase and asymmetry of protein concentration leads to narrowness of the molecular weight distribution and lowness of the average molecular weight. Using this method, 53% of hemoglobin obtained is intermolecular cross-linked with 12 molecular equivalents of glutaraldehyde. The majority of poly-hemoglobins is in the range of 128 kD to 258 kD. [ABSTRACT FROM AUTHOR]

The objective of this study was to examine the effects of polyethylene glycol (PEG) treated red blood cells (RBCs) on the microcirculation in a hamster back skin window chamber model. Donor hamster RBCs were PEGylated through an incubation with an activated PEG solution, washed, resuspended, and infused through a 10% volume top loading procedure into the carotid artery in an awake Syrian Golden hamster. Eight hamster groups were treated with activated PEG different sizes and concentrations: 0.05 mM-5 kDa PEG, 0.5 mM-5 kDa PEG, 1.1 mM-5 kDa PEG, 2.2 mM-5 kDa PEG, 22 mM-5 kDa PEG, 0.05 mM-20 kDa PEG, 0.5 mM-20 kDa PEG, and 5 mM-20 kDa PEG. Non-treated RBCs were used as control. The microvascular bed under observation was videotaped 30 min before the infusion and followed for 30 min post infusion. The diameter of individual blood vessels and blood flow velocities in selected vessels was measured. Hematocrit and hemoglobin concentration were recorded before infusion and at the end of experiment. Tissue pO2 was also monitored. Results showed the hamsters tolerated the PEGylated RBCs without apparent ill effects. No significant changes were recorded for the hematocrit, the hemoglobin concentration, the blood vessel diameters, blood flow velocities, and the interstitial partial oxygen pressure (pO2) before, during, and after the injections of PEG-RBCs (P > 0.05). Unlike most hemoglobin-based oxygen carrying compounds, which can cause vasoconstriction, the PEGylated RBCs did not produce any measurable vasoactivity. Together with the absence of rouleaux formation and the fact that PEG molecules can mask the surface antigens on RBCs, PEGylation appeared promising as a circulation enhancement treatment. [ABSTRACT FROM AUTHOR]

The article presents abstracts on medical topics which include Pegylation of Hemoglobin Cancels the Inherent Nitric Oxide Scavenging by Hemoglbin, Anesthesia Stability Associated with the Use of Perfluorocarbons During Cardiopulmonary Bypass Surgery, and Microvascular Perspective of Current Polyethylene Glycol Surface Decorated Hemoglobins: Expanding Their Boundaries with New Designs.

Hemoglobin-based blood substitute is widely studied. As the starting materials, hemoglobin (Hb) is mainly supplied from outdated human blood and animal sources. But there are many disadvantages. As the alternative source, human placenta hemoglobin (PHb) has its own advantages. We take the lead in being occupied in the research of blood substitutes based on the placenta blood. Purified PHb readily undergoes dissociation to form two dimers that are easily filtered by the kidneys and have high 02 affinity (low P50), failing to transport 02 to tissues. As a first step, to enhance PHb stability and P50, we first adopted DBBF to modify deoxyPHb leading to DBBF-αPHb. According to detection, we knew that the PHb stability was improved, 02 affinity of the PHb solution was reduced, DBBF-αPHb had the appropriate capacity of oxygen-carrying and oxygen-unloading, and DBBF-αPHb kept the biological activity well. On the whole DBBF-αPHb could be considered as a first step toward the synthesis of a potential blood substitute. [ABSTRACT FROM AUTHOR]

This study introduced a liquid-liquid extraction method designed for complete recovery of phospholipids from protein-rich samples, such as hemoglobin (Hb) solutions. In order to minimize protein denaturation and maximize lipid extraction from protein-rich samples, isopropanol was used as the major organic extraction solvent. In a wide range of the volume ratio of isopropanol to Hb solution at low temperatures, such extraction system resulted in limited protein precipitation and did not cause heme pigment contamination. The efficiency of phospholipid extraction from Hb solutions was pH-dependent, with the lowest extraction yield around neural pH and the highest extraction yield around pH 5.0. At pH 5.0, salt with concentrations up to 400 mmol/L in the samples did not increase extraction efficiency. Compared to other available methods, this method is simpler, needs significantly less organic solvent and, most importantly, consumed much less Hb, which is very expensive to make for the purpose of production of therapeutic products, hemoglobin-based oxygen carriers (HBOCs). This method is suitable not only for Hb solutions but also for other protein-rich biological samples, such as erythrocytes, plasma and liver. [ABSTRACT FROM AUTHOR]

We investigated the effect of the bovine hemoglobin based oxygen carrier HBOC-200 (Oxyglobin®) in rat aortic endothelial cells (RAEC) on the activation of heme oxygenase 1 (HO-1). RAEC were incubated in the presence of 75 μ M (G1) or 225 μ M (G2) HBOC-200. The positive control (G3) was performed by incubation with 50 μ M Hemin. For negative control (G4) cells were incubated with medium alone. G2 and G3 reached a significant increase of bilirubin concentration compared to G4. A positive HO-1 signal in the Western Blot was seen in G3 12 and 24 h after incubation. The Western Blot of G1, G2 and G4 showed no HO-1 signal. These data suggest that HBOC-200 in the applied dosage cannot induce HO-1 expression in RAEC, and may be degraded by isoenzymes at a lower level. [ABSTRACT FROM AUTHOR]

The severe adverse events observed in patients who received hemoglobin based oxygen carriers (HBOCs) were associated with the Ringer's D.L lactate resuscitative solution administered and to the excipient used in the HBOCs containing Ringer's D,L lactate and the length of storage of the preserved RBC administered to the patient at the time that the HBOCs were infused. This paper reports the quality of the red blood cells preserved in the liquid state at 4°C and that of previously frozen RBCs stored at 4°C with regard to their survival, function and safety. Severe adverse events have been observed related to the length of storage of the liquid preserved RBC stored at 4°C prior to transfusion. The current methods to preserve RBC in the liquid state in additive solutions at 4°C maintain their survival and function for only 2 weeks. The freezing of red blood cells with 40% W/V glycerol and storage at -80°C allows for storage at -80°C for 10 years and following thawing, deglycerolization and storage at 4°C in the additive solution (AS-3, Nutricel) for 2 weeks with acceptable 24 hour posttransfusion survival, less than 1% hemolysis, and moderately impaired oxygen transport function with no associated adverse events. Frozen deglycerolized RBCs are leukoreduced and contain less than 5% of residual plasma and non-plasma substances. Frozen deglycerolized RBCs are the ideal RBC product to transfuse patients receiving HBOCs. [ABSTRACT FROM AUTHOR]

The production of a fully functional bioartificial liver assist device (BLAD) would greatly enhance available treatment options for patients suffering from acute liver failure. Currently, inadequate oxygen provision to hepatocytes seeded within hollow fiber bioreactors hampers development of a viable hollow fiber-based BLAD. Experimentally, oxygen provision to primary rat hepatocytes cultured within hollow fiber bioreactors was measured, it was observed that supplementation with an oxygen carrier (bovine red blood cells at ∼ 2% human hematocrit) did not significantly improve oxygenation compared to the absence of an oxygen carrier. Therefore, an oxygen transport model of an individual hollow fiber within the bioreactor was developed and simulated (up to ∼ 10% human hematocrit) to more fully examine the effect of oxygen carrier supplementation on oxygenation within the bioreactor. The modeling analysis, supported via the experimental results, was utilized to predict optimal bioreactor operating conditions for the delivery of in vivo-like oxygen gradients to cultured hepatocytes in clinically relevant settings. [ABSTRACT FROM AUTHOR]

Polyhemoglobin (polyHb) is currently being assessed in phase III trials under various formulations. At present, none contain clotting factors or platelet substitutes to aid in hemostasis. We have prepared a novel blood substitute that is an oxygen carrier with platelet-like activity. This is formed by crosslinking fibrinogen to hemoglobin to form polyhemoglobin-fibrinogen (polyHb-Fg). This was studied and compared to polyHb for its effect on coagulation both in vitro and in vivo. In the in vitro experiments, PolyHb-Fg showed similar clotting times as whole blood, whereas polyHb showed significantly higher clotting times. This result was confirmed in in vivo experiments using an exchange transfusion rat-model. Using PolyHb, exchange transfusion of 80% or more increased the normal clotting time (1-2 mins) to > 10 mins. Partial clots formed with PolyHb did not adhere to the tubing wall. With PolyHb-Fg, a normal clotting time is maintained, even with 98% exchange transfusion. [ABSTRACT FROM AUTHOR]

Hemoglobin-based oxygen carriers (HBOCs) show potential as safe, efficacious, pre-hospital resuscitation fluids. The major criticism of HBOC-201 is its vasoactive property, attributed partially to low-molecular weight (low-MW) tetrameric/dimeric (TD) hemoglobin (Hb) in HBOC solution. Here we sought to determine whether resuscitation with decreasing concentrations of low-MW Hb component of HBOC affects immune responses in hemorrhagic swine. 28 anesthetized swine underwent a soft muscle crush and controlled hemorrhage of 55% blood volume, followed by resuscitation with HBOC containing 31%, 2%, or 0.4% low-MW Hb in four 10 ml/kg infusions at 20, 30, 45 and 60 minutes before hospital arrival at 75 minutes. IL-10, cell activation and adhesion markers and CD4:CD8 ratio remained unchanged in all 3 groups compared to baseline. Leukocyte apoptosis was equally elevated across all groups. Purification from 31% to 0.4% low-MW Hb in HBOC solution did not alter immune effects in a swine model of severe controlled hemorrhagic shock. [ABSTRACT FROM AUTHOR]

In this study, immunogenicity of human hemoglobin (hHb), bovine hemoglobin (bHb), porcine hemoglobin (pHb) and their glutaradehyde polymerized derivatives (hPolyHb, bPolyHb and bPolyHb, respectively) were compared. The nature of the dominant antigen determinants of the chemically polymerized proteins was studied. Glutaraldehyde chemical reaction enhanced the immunogenicity of the hemoglobin derivatives. In mice, the extent of the enhancement was largely comparable among hPolyHb, bPolyHb and pPolyHb. Using the methods of semi-quantitative western blotting and quantitative protein array, it was found that most of the polycloncal antibodies raised in rodents against glutaraldehyde polymerized hemoglobin derivatives of human, bovine or porcine species only weakly or did not cross-react with the hemoglobin derivatives of the other two species, indicating that hPolyHb, bPolyHb and bPolyHb vary significantly in their dominant antigen determinants, despite very high degree of identity in their primary amino acid sequences and high similarity in their three dimensional structures. [ABSTRACT FROM AUTHOR]

Isolation and purification of bovine hemoglobin (HbBv) was carried out after reaction of whole blood with carbon monoxide. Washing/centrifugation steps were used to eliminate leukocytes, platelets, and plasma proteins. Hypotonic media and ultrasound radiation were used to lyse red blood cells. Lyse by ultrasound was shown to lead to solutions at the highest concentrations in HbBv, and the least concentrations in major phospholipids contaminants. Additional purification procedures were performed to remove membrane proteins and phospholipids. In the first case, proteins were denatured by thermal treatment, and filtered. To eliminate phospholipids, liquid chromatography was used with strong anion exchangers. Purity of HbBv was evaluated by normal phase high performance liquid chromatography (HPLC), electrophoresis, and size-exclusion HPLC. [ABSTRACT FROM AUTHOR]

Hemoglobin-based oxygen carrier-201 transports oxygen and improves survival in swine with hemorrhagic shock, but has potential to be immune activating. Herein, we evaluated HBOC-201's immune effects in swine with more severe hemorrhagic shock due to soft tissue injury and 55% blood volume catheter withdrawal over 15 minutes followed by fluid resuscitation at 20 minutes with HBOC-201, Hextend, or no treatment (NON) before hospital arrival. Survival rates were similar with HBOC-201 and Hextend (p > 0.05), but were higher than in (p = 0.007). There were no significant group differences in blood cell count, percentages of leukocyte sub-populations and immunophenotype (CD4:CD8 ratio), adhesion markers expression (neutrophil CD11b; monocyte or neutrophil CD49d) and apoptosis. There was a trend to higher plasma IL-10 in HBOC-201 and groups vs. Hextend. We conclude that in swine with severe controlled HS and soft tissue injury, immune responses are similar with resuscitation with HBOC-201 and Hextend. [ABSTRACT FROM AUTHOR]

Hemoglobin vesicle (HbV), a liposomal oxygen carrier containing human hemoglobin, was intravenously infused into rats. After the infusion of saline, the HbV or empty vesicle (EV), numbers of red cells, leukocytes and platelets in peripheral blood were unchanged during the observation period of one week in addition to each time point among three groups. However, the lymphocyte ratio transiently decreased and the granulocyte ratio increased in the HbV and EV groups at 6 h after the infusion. Those changes returned to the initial value one day after the infusion and those were maintained for the subsequent observation period. No dramatic change was seen in the ratio of CD4+/CD8+ T cells. A transient decrease of the complement titer was observed three days after the infusion of HbV and EV, although the consumption of complement titer was not detected in rat serum by mixing HbV or EV in vitro, indicating that the transient decrease of complement titer in vivo was not due to the consumption of complement due to the interaction with HbV or EV. Multiple infusions of HbV caused the decrease of complement titer only after the first infusion and no allergic reaction was observed. No anaphylactic shock was observed in rats administered with EV several times, while ovalbumin (OVA) sensitized rats died with symptoms of respiratory distress after the second OVA administration. These results indicate that HbV could be administered without serious clinical symptoms or adverse reactions. [ABSTRACT FROM AUTHOR]

Despite the tremendous progress in research on hemoglobin (Hb) cellular and molecular responses, the current understanding of Hb's overall intrinsic toxicity is still limited. The complete mechanism of Hb-induced vasoconstriction has not yet been established, particularly the involvement of the renin-angiotensin system (RAS). Some studies emphasized that Hb may augment the vascular responsiveness to angiotensin (Ang)-II. It was also reported that Hb, as well as Ang-II, influences the synthesis of 8-iso prostaglandin F2 alpha, which has an impact on renal flow and possibly RAS. Hb in the presence of H2O2 gains enzymatic activity. Thus, it is possible that Hb directly and/or indirectly can activate RAS. In this study, we monitored the effect of ferrous- and ferryl-Hb, and H2O2 alone, on conversion of Ang-I to its active metabolites. The structural and immunological identity of the resulting products were evaluated by reversed phase C-18 HPLC and ELISA, respectively. Additionally, ACE-like activity of Hbs was measured spectrophotometrically by determining their ability to react with the ACE substrate, the synthetic tripeptide N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine. Results indicate that while ferrous-Hb can serve as a receptor for Ang-I, its ferryl form possesses ACE-like activity, being able to convert, within minutes, Ang-I to Ang-II, Ang-III, Ang-IV, Ang (1-7) and other unresolved fragments. H2O2 itself had a very limited hydrolyzing effect on Ang-I. Based on this study, it can be concluded that ACE-like activity of Hb with rapid formation of active angiotensins may be a contributor to the still unexplained vasoconstrictive response observed immediately after Hb administration. [ABSTRACT FROM AUTHOR]

In this study capillary zone electrophoresis (CZE) was used to analyze the poly(ethylene glycol)-modified bovine hemoglobin(PEG-bHb). The results show that CZE separated the subunit of bovine hemoglobin based on the number of PEG conjugating to the protein surface, which makes it possible to evaluate the degree of modification of hemoglobin subunit; meanwhile, it also reflected the stability of PEG attaching to hemoglobin after incubating with hydroxylamine, which makes it successful to detect the distribution of attachment site and evaluate the stability of PEG on the surface of hemoglobin. As a simple, fast and accurate method, CZE is suitable to monitor the production procedures and quality control of the final products of the PEG-bHb. [ABSTRACT FROM AUTHOR]

Intramolecular crosslink of hemoglobin tetramers solved the problem of urine elimination and short intravascular retention time of cell free hemoglobin infusion. It also produced a family of crosslinked hemoglobins with P50 between 18 and 30 mmHg. However, it did not solve the problem of MAP increases in infused animals. It was proven that extravasation of hemoglobin into interstitial fluid was responsible for MAP increases. Extravasation and the MAP increase was avoided using a hemoglobin polymer with average size near 25 MDa. In spite of a very high oxygen affinity, this polymer delivered oxygen to tissues, producing either vasodilation or vasoconstriction according to oxygen needs. It was also proven that cell free hemoglobins are more efficient than red cells in delivering oxygen to tissues. [ABSTRACT FROM AUTHOR]

The development of hexaPEGylated Hb, (SP-PEG5K)6-Hb, using the newly designed thiolation-mediated maleimide chemistry based PEGylation, has validated the concept that engineering ‘plasma volume expander’ -like properties to Hb neutralizes its vasoactivity. The high O2 affinity of hexaPEGylated Hb has been attributed to the two PEG-5K chains on its two Cys-93(β) residues. In an attempt to map the influence of the additional four PEG-5K chains of HexaPEGylated Hb on the O2 affinity, we have now investigated the influence of PEGylation of the surface amino groups alone on the subunit interface interactions and O2 affinity of Hb using rHb(βC93A). The molecular radius of PEGylated rHb(βC93A) was slightly smaller than that of (SP-PEG5K)6-Hb, and the overall site-selectivity of PEGylation in the PEGylated rHb(βC93A) at Lys-residues was comparable to that of (SP-PEG5K)6-Hb. Proton NMR studies have shown that the conjugation of the protein with PEG-5K does not have any significant influence on its subunit interface interactions. Surprisingly, the influence of PEGylation on the O2 affinity and Bohr effect of HbA and rHb(βC93A) is also nearly the same. Apparently, conjugation of PEG-chains to Lys residues of Hb by the thiolation mediated PEGylation induces unique changes in the structure of the hydration shell of Hb (layer of tightly bound water molecules), which, in turn, induces constraints in its R to T conformational transition to favor the more hydrated R-state. [ABSTRACT FROM AUTHOR]

To increase the overall size of hemoglobin (Hb), we developed a novel system of polymerization based on the complementary chemistry between sulfhydryls and maleimides. The maleimides were introduced onto the protein through N-(-maleimidobutyryloxy) succinimide, while the sulfhydryls were added using 2-iminothiolane hydrochloride (Trauts reagent). Resulting polymers showed SDS-PAGE bands with molecular weights as high as 96 kDa. Size exclusion chromatography has demonstrated species with molecular weight > 700 kDa. The flexibility of the sulfhydryl-maleimide chemistry has also allowed insertion of two antioxidant enzymes, catalase (Cat) and superoxide dismutase (SOD), into the Hb polymer. Cat was incorporated into the heavier fractions of the polymer, while SOD was found throughout the molecular weight range. [ABSTRACT FROM AUTHOR]

Natural acellular polymeric hemoglobins (Hb) provide oxygen transport and delivery within many terrestrial and marine invertebrate organisms. It has been our premise that these natural acellular Hbs may serve as models of therapeutic hemoglobin-based oxygen carriers (HBOC). Our attention has focused on the acellular Hb from the terrestrial invertebrate, Lumbricus terrestris (Lt), which possesses a unique hierarchical structure and a unique ability to function extracellularly without oxidative damage. Lumbricus Hb and Arenicola Hb are resistant to autoxidation, chemical oxidation by potassium ferricyanide, and have little or no capacity to transfer electrons to Fe+3-complexes at 37°C. An understanding of how these invertebrate acellular oxygen carriers maintain their structural integrity and redox stability in vivo is vital for the design of a safe and effective red cell substitute. We report here a positive redox potential for these giant hemoglobins that may lie at the basis for its resistance to oxidation. [ABSTRACT FROM AUTHOR]

To gain insight into the degree to which hemoglobin based oxygen carriers may replace the need for transfusion in surgical patients experiencing blood loss, a simple mathematical model was developed. This model predicts the amount of blood sparing resulting from a bolus infusion of different doses of hemoglobin solution as a function of circulating hemoglobin half-life and degree of erythropoesis enhancement subsequent to treatment. The results of this analysis are consistent with published clinical data and imply that blood sparing increases with increasing oxygen carrier dose and half-life, as well as increasing levels of erythropoesis enhancement. The analysis also predicts that the total circulating hemoglobin content in patients infused with HBOC solutions may reach a minimum value up to ten days after treatment. [ABSTRACT FROM AUTHOR]

This paper describes the approaches we have taken to construct a) mutant hemoglobins with different oxygen affinities, and b) mutant hemoglobins and myoglobins that polymerize to high molecular weight aggregates in an effort to prevent extravasation and the associated vasoactivity. In vivo testing indicates that exchange transfusion of polymeric hemoglobins in mice does not result in vasoactivity and that polymeric hemoglobins are effective oxygen carriers to ischemic tissues irrespective of their oxygen affinity and cooperativity. [ABSTRACT FROM AUTHOR]

Hemoglobin-vesicles (HbV) or liposome-encapsulated Hb are artificial oxygen carriers. Our previous studies of the bolus infusion of HbV into Wistar rats showed that HbV was captured by the reticuloendothelial system from the blood stream and degraded completely with no deteriorative effect for 2 weeks. However, one authority on artificial organs research suggested conducting a one-year observation because he experienced, with one lipid-emulsified perfluorocarbon (PFC), that rats died within one year from a pulmonary abnormality after receiving the PFC emulsion due to the unstable dispersion state (personal communication). We thought this would never happen for HbV because the dispersion state of HbV is stable with PEG-modification. To confirm this, we made one-year observations after HbV infusion as suggested. Five male Wistar rats intravenously received 20 ml/kg HbV suspended in saline ([Hb] = 10 g/dL). They were housed in separated cages and provided with food and water ad libitum. All rats survived one year, and were apparently healthy. Their body weights (821±75 g) reflected obesity from their confinement in small cages. No histopathological abnormality was found in the lung. Plasma biochemical analyses showed overall normal organ functions. In our previous report, plasma lipid levels increased transiently at 1 or 2 days; then they reverted to the control level at 7 days. One year later, the rats showed much higher plasma lipid levels, a symptom of hyperlipidemia that is attributable to obesity and aging. It seemed the transient increases at the early days had no impact compared with the levels of hyperlipemia of the old rats. [ABSTRACT FROM AUTHOR]