Hemoglobin-based Oxygen Carriers Research Articles

Hemoglobin Vesicles prolong the time to circulatory collapse in rats during apnea

BackgroundHemoglobin vesicles (HbV) are hemoglobin-based oxygen carriers manufactured by liposome encapsulation of hemoglobin molecules. We hypothesised that the infusion of oxygenated HbV could prolong the time to circulatory collapse during apnea in rats.MethodsTwenty-four Sprague-Dawley rats were randomly divided into four groups (Air, Oxy, NS and HbV). The rats were anaesthetized with isoflurane and the trachea was intubated using 14-gauge intravenous catheters. Rats in the Air group were mechanically ventilated with 1.5% isoflurane in room air, and those in other groups received 1.5% isoflurane in 100% oxygen. Mechanical ventilation was withdrawn 1 min after the administration of rocuronium bromide to induce apnea. After 30 s, 6 mL saline and HbV boluses were infused at a rate of 0.1 mL/s in the NS and HbV groups, respectively. Circulatory collapse was defined as a pulse pressure < 20 mmHg and the time to reach this point (PP20) was compared between the groups. The results were analysed via a one-way analysis of variance and post-hoc Holm–Sidak test.ResultsPP20 times were 30.4 ± 4.2 s, 67.5 ± 9.7 s, 95 ± 17.3 s and 135 ± 38.2 s for the Air (ventilated in room air with no fluid bolus), Oxy (ventilated with 100% oxygen with no fluid bolus), NS (ventilated with 100% oxygen with a normal saline bolus), and HbV (ventilated in 100% oxygen with an HbV bolus) groups, respectively, and differed significantly between the four groups (P = 0.0001). The PP20 times in the HbV group were significantly greater than in the Air (P = 0.0001), Oxy (P = 0.007) and NS (P = 0.04) groups.ConclusionInfusion of oxygenated HbV prolongs the time to circulatory collapse during apnea in rats.

Bloodless reperfusion with the oxygen carrier HBOC-201 in acute myocardial infarction: a novel platform for cardioprotective probes delivery.

Reperfusion, despite being required for myocardial salvage, is associated with additional injury. We hypothesize that infarct size (IS) will be reduced by a period of bloodless reperfusion with hemoglobin-based oxygen carriers (HBOC) before blood-flow restoration. In the pig model, we first characterized the impact of intracoronary perfusion with a fixed volume (600ml) of a pre-oxygenated acellular HBOC, HBOC-201, on the healthy myocardium. HBOC-201 was administered through the lumen of the angioplasty balloon (i.e., distal to the occlusion site) immediately after onset of coronary occlusion at 1, 0.7, 0.4, or 0.2ml/kg/min for 12, 17, 30, and 60min, respectively, followed by blood-flow restoration. Outcome measures were systemic hemodynamics and LV performance assessed by the state-of-the-art cardiac magnetic resonance (CMR) imaging. The best performing HBOC-201 perfusion strategies were then tested for their impact on LV performance during myocardial infarction, in pigs subjected to 45min mid-left anterior descending (LAD) coronary occlusion.At the end of the ischemia duration, pigs were randomized to regular reperfusion (blood-only reperfusion) vs. bloodless reperfusion (perfusion with pre-oxygenated HBOC-201 distal to the occlusion site), followed by blood-flow restoration. Hemodynamics and CMR-measured LV performance were assessed at 7- and 45-day follow-up. In modifications of the HBOC-201 procedure, glucose and insulin were included to support cardiac metabolism. A total of 66 pigs were included in this study. Twenty healthy pigs (5 per infusion protocol) were used in the study of healthy myocardium. Intracoronary administration of HBOC-201 (600ml) at varying rates, including a flow of 0.4ml/kg/min (corresponding to a maximum perfusion time of 30min), did not damage the healthy myocardium. Slower perfusion (longer infusion time) was associated with permanent LV dysfunction and myocardial necrosis. A total of 46 pigs underwent MI induction. Compared with regular reperfusion, bloodless reperfusion with pre-oxygenated HBOC-201 alone increased IS. This effect was reversed by enrichment of pre-oxygenated HBOC-201 solution with glucose and insulin, resulting in no increase in IS or worsening of long-term ventricular function despite further delaying restoration of blood flow in the LAD. Bloodless reperfusion with a pre-oxygenated HBOC-201 solution supplemented with glucose and insulin is feasible and safe, but did not reduce infarct size. This strategy could be, however, used to deliver agents to the myocardium to treat or prevent ischemia/reperfusion injury before blood-flow restoration.

Normalization of hemoglobin-based oxygen carrier-201 induced vasoconstriction: targeting nitric oxide and endothelin.

Hemoglobin-based oxygen carrier (HBOC)-201 is a cell-free modified hemoglobin solution potentially facilitating oxygen uptake and delivery in cardiovascular disorders and hemorrhagic shock. Clinical use has been hampered by vasoconstriction in the systemic and pulmonary beds. Therefore, we aimed to 1) determine the possibility of counteracting HBOC-201-induced pressor effects with either adenosine (ADO) or nitroglycerin (NTG); 2) assess the potential roles of nitric oxide (NO) scavenging, reactive oxygen species (ROS), and endothelin (ET) in mediating the observed vasoconstriction; and 3) compare these effects in resting and exercising swine. Chronically instrumented swine were studied at rest and during exercise after administration of HBOC-201 alone or in combination with ADO. The role of NO was assessed by supplementation with NTG or administration of the eNOS inhibitor Nω-nitro-l-arginine. Alternative vasoactive pathways were investigated via intravenous administration of the ETA/ETB receptor blocker tezosentan or a mixture of ROS scavengers. The systemic and to a lesser extent the pulmonary pressor effects of HBOC-201 could be counteracted by ADO; however, dosage titration was very important to avoid systemic hypotension. Similarly, supplementation of NO with NTG negated the pressor effects but also required titration of the dose. The pressor response to HBOC-201 was reduced after eNOS inhibition and abolished by simultaneous ETA/ETB receptor blockade, while ROS scavenging had no effect. In conclusion, the pressor response to HBOC-201 is mediated by vasoconstriction due to NO scavenging and production of ET. Further research should explore the effect of longer-acting ET receptor blockers to counteract the side effect of hemoglobin-based oxygen carriers.NEW & NOTEWORTHY Hemoglobin-based oxygen carrier (HBOC)-201 can disrupt hemodynamic homeostasis, mimicking some aspects of endothelial dysfunction, resulting in elevated systemic and pulmonary blood pressures. HBOC-201-induced vasoconstriction is mediated by scavenging nitric oxide (NO) and by upregulating endothelin (ET) production. Pressor effects can be prevented by adjuvant treatment with NO donors or direct vasodilators, such as nitroglycerin or adenosine, but dosages must be carefully monitored to avoid hypotension. However, hemodynamic normalization is more easily achieved via administration of an ET receptor blocker.

Application of HBOCs electrophoretic method to detect a new blood substitute derived from the giant extracellular haemoglobin of lugworm.

Manipulation of blood and blood components is prohibited in sports by the World Anti-Doping Agency (WADA). This includes the use of blood substitutes to increase oxygen transport, like haemoglobin-based oxygen carriers (HBOCs), which are compounds derived from haemoglobin. Despite their medical interest, the first generation of HBOCs had serious adverse effects and was abandoned. However, new studies are now exploiting the properties of marine worm haemoglobins, which circulate as giant extracellular complexes with high oxygen-binding capacities. HEMOXYCarrier® (HC), developed by Hemarina, is one of the most advanced and promising HBOCs, and HC may become a tempting doping tool for athletes in the future. Here, HC detection in plasma/serum was evaluated with the method used to detect the first HBOCs, based on electrophoresis and heme peroxidase properties. An HC-derived product was identified in human plasma up to 72 h after in vitro incubation at 37 °C. HC degradation also induced methemalbumin formation. After injecting HC at the effective dose of 200 mg/kg into mice, the HC-derived product was detected only for a few hours and no accumulation of methemalbumin was observed. Due to this limited detection window in vivo, measuring specific worm globin degradation products by mass spectrometry might be an alternative for future anti-doping analyses. Copyright © 2016 John Wiley & Sons, Ltd.

Hemoglobin-Based Oxygen Carrier (HBOC) Development in Trauma: Previous Regulatory Challenges, Lessons Learned, and a Path Forward.

Historically, hemoglobin-based oxygen carriers (HBOCs) were being developed as "blood substitutes," despite their transient circulatory half-life (~ 24 h) vs. transfused red blood cells (RBCs). More recently, HBOC commercial development focused on "oxygen therapeutic" indications to provide a temporary oxygenation bridge until medical or surgical interventions (including RBC transfusion, if required) can be initiated. This included the early trauma trials with HemAssist ® (BAXTER), Hemopure ® (BIOPURE) and PolyHeme ® (NORTHFIELD) for resuscitating hypotensive shock. These trials all failed due to safety concerns (e.g., cardiac events, mortality) and certain protocol design limitations. In 2008 the Food and Drug Administration (FDA) put all HBOC trials in the US on clinical hold due to the unfavorable benefit:risk profile demonstrated by various HBOCs in different clinical studies in a meta-analysis published by Natanson et al. (2008). During standard resuscitation in trauma, organ dysfunction and failure can occur due to ischemia in critical tissues, which can be detected by the degree of lactic acidosis. SANGART'S Phase 2 trauma program with MP4OX therefore added lactate >5 mmol/L as an inclusion criterion to enroll patients who had lost sufficient blood to cause a tissue oxygen debt. This was key to the successful conduct of their Phase 2 program (ex-US, from 2009 to 2012) to evaluate MP4OX as an adjunct to standard fluid resuscitation and transfusion of RBCs. In 2013, SANGART shared their Phase 2b results with the FDA, and succeeded in getting the FDA to agree that a planned Phase 2c higher dose comparison study of MP4OX in trauma could include clinical sites in the US. Unfortunately, SANGART failed to secure new funding and was forced to terminate development and operations in Dec 2013, even though a regulatory path forward with FDA approval to proceed in trauma had been achieved.

Characterization of Protein-Protein Interactions in Recombinant Hemoglobin Producing Escherichia coli Cells Using Molecularly Imprinted Polymers.

The worldwide blood shortage has generated demands for alternatives to transfusible human blood. One such important option is based on recombinant hemoglobin-based oxygen carriers (rHBOCs). Most efforts have been focused on various E. coli based production systems. One of the key challenges in these systems is to devise an efficient and economical protein production strategy involving selection of suitable host cell and Hb variant, growth conditions and media engineering. Hb also influences the heterologous host cell metabolism and therefore the identification of modified protein-protein interactions is critical for optimizing Hb production. In this study, molecularly imprinted polymers (MIPs) directed against Hb were used to identify the human Hb protein interaction network in E. coli. One E. coli host protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), interacted strongly with Hb, especially fetal Hb (HbF).

Searching For the Optimal Fluid to Restore Microcirculatory Flow Dynamics After Haemorrhagic Shock: A Systematic Review of Preclinical Studies.

Increased microcirculatory flow and perfusion has been reported to improve clinical outcomes following shock. The optimal resuscitation fluid to restore the flow dynamics of the microcirculation is unknown. This review summarizes the preclinical literature to inform the direction and most important hypotheses for future clinical interventional studies. Standard systematic review methodology was utilized, and registered with the Collaborative Approach to Meta Analysis and Review of Animal Data from Experimental Studies (CAMARADES). Medline and Embase (via OVID SP) and SCOPUS were searched for all preclinical studies of haemorrhagic shock that compared fluid resuscitation of any kind (e.g., blood products, crystalloids, colloids, or haemoglobin based oxygen carriers) to another fluid or haemorrhage only, and reported at least one microcirculatory physical endpoint (such as flow rate, velocity, vessel diameter, functional capillary density, or glycocalyx thickness). Risk of bias was assessed using the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) tool. Translatability was also assessed for each study based on the most common recommendations. There were 3,103 potential studies of interest, of which 71 studies fulfilled all eligibility criteria. There were 62 rodent, 5 canine, and 4 porcine studies. Flow rate, velocity, and vessel diameter were the most commonly reported endpoints. Studies reported the importance of the presence of haemoglobin, as well as osmotic potential and viscosity in providing optimal restoration of microcirculatory flow dynamics. Others reported the restoration of the endothelial glycocalyx and attenuation of inflammation as important properties for the choice of fluid. All studies were at potential risk of bias due to unclear randomization, concealment, and blinding. There were important threats to translatability for all studies. The ideal resuscitation fluid for restoration of the microcirculation following haemorrhagic shock is likely to contain a preparation of haemoglobin, favor higher oncotic potential, and viscosity, protect and reconstitute the endothelium, and attenuate inflammation. These hypotheses that are derived from preclinical research warrant further exploration in the clinical context.

Hemoglobin extravasation in the brain of rats exchange-transfused with hemoglobin-based oxygen carriers

Haemoglobin (Hb)-based oxygen carriers are under consideration as oxygen therapeutics. Their effect on apoptosis is critical, because the onset of pro-apoptotic pathways may lead to tissue damage. MP4OX, a polyethylene glycol-conjugated human Hb preserves the baseline level of neuron apoptosis with respect to sham. Here we develop a method for measuring Hb extravasation in brain. We exchange transfused rats by haemorrhaging 50% of their blood with simultaneous, isovolemic replacement with Hextend (negative control), MP4OX, or αα-cross-linked Hb. Animals were sacrificed 2 h after transfusion, brain tissue was harvested and processed for double-staining immunofluorescence, whereby Hb ? chain and NeuN (a neuron protein) were stained and quantitated. Whereas Hextend did not induce Hb extravasation, in both MP4OX and ??Hb brains Hb molecules were detected outside neurons. The level of extravasated Hb chains was > 3-fold higher in Hb compared to MP4OX. Western blot analysis revealed that the expression levels of protein related to redox imbalance (e.g., Nrf2, iNOS and ERK phosphorylation) were higher in ααHb than MP4OX. In conclusions, higher Hb extravasation in ααHb than MP4OX induces redox imbalance, which causes higher anti-oxidant response. Whereas Nrf2 response may be considered protective, iNOS response appears damaging.

Beneficial effects of novel cross-linked hemoglobin YQ23 on hemorrhagic shock in rats and pigs

BackgroundTo overcome the problems of previously reported hemoglobin-based oxygen carriers, we developed a stabilized nonpolymeric cross-linked tetrameric hemoglobin solution (YQ23). The aims of this study were to investigate the oxygen carrying and releasing properties of this novel hemoglobin-based oxygen carrier and to determine whether it has beneficial effects for hemorrhagic shock. MethodsUsing a hemorrhagic shock model in Sprague–Dawley rats and mini-pigs, we tested the effects of infusing 0.1, 0.3, and 0.5 g/kg YQ23 on animal survival, tissue oxygen delivery (DO2) and consumption (VO2), hemodynamics parameters, and liver, renal, and cardiac function. ResultsYQ23 infusion increased the survival rate of rats and pigs with severe hemorrhagic shock in a dose-dependent manner. Moreover, it improved the hemodynamic parameters, cardiac output, DO2 and VO2, and the mitochondrial respiratory function of vital organs. Among the three doses of YQ23, 0.5 gHb/kg YQ23 achieved a similar beneficial effect as whole blood. ConclusionsThis study indicated that the novel cross-linked tetrameric hemoglobin YQ23 has good oxygen carrying and releasing properties and exhibits beneficial effects on hemorrhagic shock in rats and pigs by improving the oxygen carrying and delivery function of blood, which maintains organ function.

Oxygen carrying capability evaluation based on direct electrochemistry of highly loaded hemoglobin spheres

Hemoglobin-based oxygen carriers (HBOCs) are widely researched as blood substitute and mainly produced based on templates. As reported, their optimal size should be preferably less than 1µm and more than 100nm, but porous calcium carbonate (CaCO3) is the most common template to synthesis HBOCs and their average size is larger than 3µm. Therefore, in this study, we employed manganese carbonate (MnCO3) instead of CaCO3 for the production of much smaller multilayer hemoglobin (Hb) sphere with glutaraldehyde (GA) as the crosslinking agent. According to our previous work, the obtained {Hb/GA}n nanospheres were also immobilized on chitosan modified glassy carbon electrode (GCE) to evaluate their oxygen-carrying ability with electrochemical techniques. By comparison, {Hb/GA}5 demonstrates a higher sensitivity to oxygen, stronger carrying-oxygen ability and more stable properties than {Hb/GA}1. The present work suggests potential applications of HBOCs as blood substitute.

Addressing the unmet need of life-threatening anemia with hemoglobin-based oxygen carriers.

Addressing the unmet need of life-threatening anemia with hemoglobin-based oxygen carriers.

Machine Perfusion of Porcine Livers with Oxygen-Carrying Solution Results in Reprogramming of Dynamic Inflammation Networks.

Background: Ex vivo machine perfusion (MP) can better preserve organs for transplantation. We have recently reported on the first application of an MP protocol in which liver allografts were fully oxygenated, under dual pressures and subnormothermic conditions, with a new hemoglobin-based oxygen carrier (HBOC) solution specifically developed for ex vivo utilization. In those studies, MP improved organ function post-operatively and reduced inflammation in porcine livers. Herein, we sought to refine our knowledge regarding the impact of MP by defining dynamic networks of inflammation in both tissue and perfusate.Methods: Porcine liver allografts were preserved either with MP (n = 6) or with cold static preservation (CSP; n = 6), then transplanted orthotopically after 9 h of preservation. Fourteen inflammatory mediators were measured in both tissue and perfusate during liver preservation at multiple time points, and analyzed using Dynamic Bayesian Network (DyBN) inference to define feedback interactions, as well as Dynamic Network Analysis (DyNA) to define the time-dependent development of inflammation networks.Results: Network analyses of tissue and perfusate suggested an NLRP3 inflammasome-regulated response in both treatment groups, driven by the pro-inflammatory cytokine interleukin (IL)-18 and the anti-inflammatory mediator IL-1 receptor antagonist (IL-1RA). Both DyBN and DyNA suggested a reduced role of IL-18 and increased role of IL-1RA with MP, along with increased liver damage with CSP. DyNA also suggested divergent progression of responses over the 9 h preservation time, with CSP leading to a stable pattern of IL-18-induced liver damage and MP leading to a resolution of the pro-inflammatory response. These results were consistent with prior clinical, biochemical, and histological findings after liver transplantation.Conclusion: Our results suggest that analysis of dynamic inflammation networks in the setting of liver preservation may identify novel diagnostic and therapeutic modalities.

Long-Term Stored Hemoglobin-Vesicles, a Cellular Type of Hemoglobin-Based Oxygen Carrier, Has Resuscitative Effects Comparable to That for Fresh Red Blood Cells in a Rat Model with Massive Hemorrhage without Post-Transfusion Lung Injury.

Hemoglobin-vesicles (HbV), encapsulating highly concentrated human hemoglobin in liposomes, were developed as a substitute for red blood cells (RBC) and their safety and efficacy in transfusion therapy has been confirmed in previous studies. Although HbV suspensions are structurally and physicochemically stabile for least 1-year at room temperature, based on in vitro experiments, the issue of whether the use of long-term stored HbV after a massive hemorrhage can be effective in resuscitations without adverse, post-transfusion effects remains to be clarified. We report herein on a comparison of the systemic response and the induction of organ injuries in hemorrhagic shock model rats resuscitated using 1-year-stored HbV, freshly packed RBC (PRBC-0) and by 28-day-stored packed RBC (PRBC-28). The six-hour mortality after resuscitation was not significantly different among the groups. Arterial blood pressure and blood gas parameters revealed that, using HbV, recovery from the shock state was comparable to that when PRBC-0 was used. Although no significant change was observed in serum parameters reflecting liver and kidney injuries at 6 hours after resuscitation among the three resuscitation groups, results based on Evans Blue and protein leakage in bronchoalveolar lavage fluid, the lung wet/dry weight ratio and histopathological findings indicated that HbV as well as PRBC-0 was less predisposed to result in a post-transfusion lung injury than PRBC-28, as evidenced by low levels of myeloperoxidase accumulation and subsequent oxidative damage in the lung. The findings reported herein indicate that 1-year-stored HbV can effectively function as a resuscitative fluid without the induction of post-transfused lung injury and that it is comparable to fresh PRBC, suggesting that HbV is a promising RBC substitute with a long shelf-life.

Carbon Monoxide-Saturated Hemoglobin-Based Oxygen Carriers Attenuate High-Altitude-Induced Cardiac Injury by Amelioration of the Inflammation Response and Mitochondrial Oxidative Damage

Objective: To investigate the therapeutic effect of carbon monoxide (CO) on high-altitude hypoxia-induced cardiac damage. Methods: Forty male C57BL/6 mice were randomly divided into 4 groups. The mice were exposed to normoxia or simulated 5,500-meter high-altitude hypoxia in a hypobaric chamber for 7 days. During the first 3 days, the mice were pretreated with CO-saturated hemoglobin (Hb)-based oxygen carrier (CO-HBOC), oxygen-saturated hemoglobin-based oxygen carrier (O₂-HBOC) at a dose of 0.3 g Hb/kg/day or an equivalent volume of saline. The in vivo left ventricle function, cardiac enzyme release, histopathological changes, apoptosis and inflammation were also measured. Results: High-altitude hypoxia induced significant cardiac damage, as demonstrated by impaired cardiac function and increased proapoptotic, proinflammatory and pro-oxidant markers. Pretreatment with CO-HBOC significantly improved cardiac performance, reduced cardiac enzyme release and limited myocardial apoptosis. The increased inflammatory response was also suppressed. In addition to the preserved mitochondrial structure, hypobaric hypoxia-induced mitochondrial oxidative damage was remarkably attenuated. Moreover, these antiapoptotic and antioxidative effects were accompanied by an upregulated phosphorylation of Akt, ERK and STAT3. Conclusion: This study demonstrated that CO-HBOC provides a promising protective effect on high-altitude hypoxia-induced myocardial injury, which is mediated by the inhibition of inflammation and mitochondrial oxidative damage.

Management of the Jehovah's Witness in Obstetrics and Gynecology: A Comprehensive Medical, Ethical, and Legal Approach.

Obstetricians and gynecologists frequently deal with hemorrhage so they should be familiar with management of patients who refuse blood transfusion. Although there are some reports in the literature about management of Jehovah's Witness patients in obstetrics and gynecology, most of them are case reports, and a comprehensive review about these patients including ethicolegal perspective is lacking. This review outlines the medical, ethical, and legal implications of management of Jehovah's Witness patients in obstetrical and gynecological settings. A search of published literature using PubMed, Ovid Medline, EMBASE, and Cochrane databases was conducted about physiology of oxygen delivery and response to tissue hypoxia, mortality rates at certain hemoglobin levels, medical management options for anemic patients who refuse blood transfusion, and ethical/legal considerations in Jehovah's Witness patients. Early diagnosis of anemia and immediate initiation of therapy are essential in patients who refuse blood transfusion. Medical management options include iron supplementation and erythropoietin. There are also some promising therapies that are in development such as antihepcidin antibodies and hemoglobin-based oxygen carriers. Options to decrease blood loss include antifibrinolytics, desmopressin, recombinant factor VII, and factor concentrates. When surgery is the only option, every effort should be made to pursue minimally invasive approaches. All obstetricians and gynecologists should be familiar with alternatives and "less invasive" options for patients who refuse blood transfusions.

Development of “Inside‐Out” PEGylated Crosslinked Hemoglobin Polymers: A Novel Hemoglobin‐Based Oxygen Carrier (HBOC)

Development of “Inside‐Out” PEGylated Crosslinked Hemoglobin Polymers: A Novel Hemoglobin‐Based Oxygen Carrier (HBOC)

Normothermic machine perfusion of donor livers using a novel hemoglobin based oxygen carrier solution, eliminating the need for human blood products

Normothermic machine perfusion of donor livers using a novel hemoglobin based oxygen carrier solution, eliminating the need for human blood products

Determination of methemoglobin and hemoglobin levels in small volume samples

Background/aims Hemoglobin-based oxygen carriers (HBOCs) have been previously studied as resuscitation fluids. Due to HBOCs specific molecular conformation, hemoglobin (Hb) and methemoglobin (MetHb) determination is not always possible with automated apparatus. A practical technique was designed that allows simultaneous reading of MetHb and Hb in small volume samples. Methods A spectrophotometric method for measuring low levels of MetHb and Hb from limited volume samples was developed using a 96-well-plate by downsizing the Evelyn-Malloy and Drabkin methods. Either blood or buffer samples were spiked with one of five HBOCs (HBOC-201, M101, MP4CO-NP, Sanguinate and Oxyvita C). After treatment with cyanides, the samples were read at 540, 630, and 680 nm, and Hb and MetHb results were compared to certificate-of-analysis. Results Hb levels ranging from 0.2 to 2.8 g/dl were detected accurately with the 96-well-plate method with HBOC-201. Similarly, this method accurately measured Hb from either plasma or buffer samples containing any of the HBOCs. The MetHb plasma samples with HBOC-201 were also in agreement with ABL results (R = 0.99719). MetHb from all HBOCs in buffer measured with this method was comparable to reference but the accuracy was compromised for HBOCs in blood. Conclusions A useful 96-well-plate method of measuring HBOCs’ Hb was designed for small-volume plasma samples. It was accurate for measuring MetHb from samples, that contained M101, MP4CO-NP, Sanguinate, and Oxyvita C diluted in buffer. This well-plate method allows reading of batch samples, multiple replicates, and using small volumes to accommodate limited animal blood collection which would not be otherwise detected by automated instrumentation.

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.

Processing of ferulic acid modified hemoglobin

The application of hemoglobin-based oxygen carriers (HBOCs) remains stagnant because a large number of reactive oxygen species (ROS) will be produced in organisms when HBOCs are used. Ferulic acid (FA) has a known effect on quenching oxygen free radicals and relieving vasospasm. Thus a kind of FA modified hemoglobin (BAEGF-Hb) is expected to produce the anti-oxidation effect on HBOCs spontaneously, and reduce toxicity. Isoelectric focusing electrophoresis, UV–vis wavelength scanning, and oxygen affinity curve showed that hemoglobin (Hb) was successfully modified and this modification had no effect on the structure of Hb and P50. The modification degree of Hb is 4.