The widespread adoption of high-calorie, high-fat, high-sucrose diets (HFHSD) has become a global health concern, particularly due to their association with cardiovascular diseases and metabolic disorders. These comorbidities increase susceptibility to severe outcomes from viral infections and trauma, with trauma-related incidents significantly contributing to global mortality rates. This context underscores the critical need for a reliable blood supply. Recent research has focused on high molecular weight (MW) polymerized human hemoglobin (PolyhHb) as a promising alternative to red blood cells (RBCs), showing encouraging outcomes in previous studies. Given the overlap of metabolic disorders and trauma-related health issues, it is crucial to assess the potential toxicity of PolyhHb transfusions, particularly in models that represent these vulnerable populations. This study evaluated the effects of PolyhHb exchange transfusion in guinea pigs that had developed metabolic disorders due to a 12-week HFHSD regimen. The guinea pigs, underwent a 20 % blood volume exchange transfusion with either PolyhHb or the lower molecular weight polymerized bovine hemoglobin, Oxyglobin. Results revealed that both PolyhHb and Oxyglobin transfusions led to liver damage, with a more pronounced effect observed in HFHSD-fed animals. Additionally, markers of cardiac dysfunction indicated signs of cardiac injury in both the HFHSD and normal diet groups following the Oxyglobin transfusion. This study highlights how pre-existing metabolic disorders can exacerbate the potential side effects of hemoglobin-based oxygen carriers (HBOCs). Importantly, the newer generation of high MW PolyhHb showed lower cardiac toxicity compared to the earlier generation low MW PolyhHb, known as Oxyglobin, even in models with pre-existing endothelial and metabolic challenges.

For the past thirty years, hemoglobin-based oxygen carriers (HBOCs) have been under development as a red blood cell substitute. Side-effects such as vasoconstriction, oxidative injury, and cardiac toxicity have prevented clinical approval of HBOCs. Recently, high molecular weight (MW) polymerized human hemoglobin (PolyhHb) has shown positive results in rats. Studies have demonstrated that high MW PolyhHb increased O2 delivery, with minimal effects on blood pressure, without vasoconstriction, and devoid of toxicity. In this study, we used guinea pigs to evaluate the efficacy and safety of high MW PolyhHb, since like humans guinea pigs cannot produce endogenous ascorbic acid, which limits the capacity of both species to deal with oxidative stress. Hence, this study evaluated the efficacy and safety of resuscitation from severe hemorrhagic shock with high MW PolyhHb, fresh blood, and blood stored for 2 weeks. Animals were randomly assigned to each experimental group, and hemorrhage was induced by the withdrawal of 40% of the blood volume (BV, estimated as 7.5% of body weight) from the carotid artery catheter. Hypovolemic shock was maintained for 50 min. Resuscitation was implemented by infusing 25% of the animal's BV with the different treatments. Hemodynamics, blood gases, total hemoglobin, and lactate were not different before hemorrhage and during shock between groups. The hematocrit was lower for the PolyhHb group compared to the fresh and stored blood groups after resuscitation. Resuscitation with stored blood had lower blood pressure compared to fresh blood at 2 h. There was no difference in mean arterial pressure between groups at 24 h. Resuscitation with PolyhHb was not different from fresh blood for most parameters. Resuscitation with PolyhHb did not show any remarkable change in liver injury, inflammation, or cardiac damage. Resuscitation with stored blood showed changes in liver function and inflammation, but no kidney injury or systemic inflammation. Resuscitation with stored blood after 24 h displayed sympathetic hyper-activation and signs of cardiac injury. These results suggest that PolyhHb is an effective resuscitation alternative to blood. The decreased toxicities in terms of cardiac injury markers, vital organ function, and inflammation following PolyhHb resuscitation in guinea pigs indicate a favorable safety profile. These results are promising and support future studies with this new generation of PolyhHb as alternative to blood when blood is unavailable.

Chronic pharmacologically increased hemoglobin affinity for oxygen in sickle cell disease mice alleviated hematological consequences of sickle cell disease, increasing RBC half-life, hematocrit, and hemoglobin concentration, while also decreasing reticulocyte count. Additionally, chronically increased hemoglobin affinity for oxygen significantly improved survival as well as cortical tissue oxygenation in sickle cell disease mice during hypoxia, suggesting that oxygen delivery and utilization is improved by increased hemoglobin affinity for oxygen.

BACKGROUND:Hemoglobin (Hb)-based oxygen (O2 ) carriers (HBOCs) are being developed as alternatives to red blood cells and blood when these products are unavailable. Clinical trials of previous HBOC generations revealed side effects, including hypertension and vasoconstriction, that were not observed in preclinical studies. Large molecular weight (MW) polymerized bovine Hb (PolybHb) represents a new class of HBOC with promising results. We evaluated the safety profile of PolybHb after an exchange transfusion (ET) in guinea pigs (GPs). This study compares changes in indices of cardiac, inflammatory, and organ function after ET with high (R-state) and low (T-state) O2 affinity PolybHb with high MW.STUDY DESIGN AND METHODS:Guinea pigs underwent a 20% ET with PolybHb. To assess the implication of PolybHb ET on the microcirculation, hamsters instrumented with a dorsal window chamber were subjected to a similar volume ET.RESULTS:T and R-state PolybHb did not induce significant alterations in cardiac function. T-state PolybHb induced mild vasoconstriction shortly after transfusion, while R-state did not have acute effects on microvascular tone.CONCLUSION:Large MW PolybHbs were found to be safe and efficacious in increasing O2 carrying capacity and the O2 affinity of the PolybHb did not affect O2 delivery or extraction by tissues in relevant preclinical models. In conclusion, these results suggest that both T-state and R-state PolybHb are safe and do not impair O2 delivery. The results are encouraging and support further evaluation of high MW PolybHbs and their future feasibility compared to allogenic blood in a trauma model.

Easy access to high-calorie and fat-dense fast food has resulted in unhealthy dietary and lifestyle changes worldwide, which affects both developed and developing economies. This predisposes populations to a considerable number of metabolic and inflammatory conditions, such as diabetes, nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease (CVD). Guinea pigs have been proposed as a model to study high-fat diet-induced metabolic disease due to their similar antioxidant metabolism and lipid profile to humans, and their susceptibility to atherosclerosis and endothelial disease. This study aims to evaluate cardiovascular and metabolic disorders induced by high-fat high-sucrose diet (HFHSD) in guinea pigs. Two to three-week-old male guinea pigs were fed a normal diet (ND) or HFHSD for 12 wk. Guinea pigs fed a HFHSD developed glucose intolerance, dyslipidemia, and liver, cardiac, and kidney damage. However, hypertension, dysautonomia, endothelial disease, and obesity were absent in these HFHSD guinea pigs. Taken together, these results show that guinea pigs fed a HFHSD are a nonobese model of metabolic disorders, resulting in important cardiac damage. Moreover, our findings suggest that NAFLD may be an important risk factor for diet-induced CVD. NEW & NOTEWORTHY In this study, we show a new animal model for diet-induced disease metabolic disorders without obesity in guinea pigs. Moreover, results suggest a strong relation between liver disease and increased cardiovascular risks.

Hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) have been developed as an alternative to red blood cells (RBCs) for use in transfusion medicine. HBOCs have many benefits over RBCs; however, previous generations of HBOCs failed in clinical trials due to unanticipated cardiotoxicity. These problems likely originated from vasoconstriction, hypertension, oxidative stress, and the presence of low-molecular-weight (MW) Hb species in the HBOC formulation. Therefore, the objective of this study is to compare the toxicity of small-MW Polymerized bovine Hb (SPolyHb) to large-MW Polymerized bovine Hb (LPolyHb) in guinea pigs, since they lack the ability to synthesize vitamin C and are more sensitive to oxidative stress than other preclinical animal models. The two PolyHbs used in this study have similar molecular diameters (72 and 69 nm, respectively), but the SPolyHb included approximately 15% Hb polymers with MW below 256 kDa, which were significantly removed from LPolyHb. Solutions were injected as a hypervolemic (topload) infusion of 10% of the blood volume into animals. SPolyHb caused a 50% elevation in mean arterial pressure (MAP) from the baseline, while LPolyHb caused only a small increase in MAP. Both PolyHbs also increased markers of organ damage and tissue and systemic inflammation compared to controls. SPolyHb caused significant changes in tissue function and vital organ toxicity markers compared to LPolyHb, specifically markers related to kidney, liver, and lung injury and systemic inflammation and iron transport by the reticuloendothelial system. LPolyHb had a longer half-life than SPolyHb, which correlates with observations made in the reticuloendothelial and iron transport systems. These studies indicate that the molecular size of PolyHb determines vasoactivity, circulation time, mechanism of elimination, toxicity, and inflammation induced by its infusion.

The need for alternatives to allogeneic red blood cells (RBCs) for transfusion medicine has been recognized for more than a century. Large molecular size polymerized human hemoglobin (Hb) (PolyhHb) is a Hb based oxygen (O2) carrier (HBOC) recently evaluated with promising results in efficient O2 delivery and minimal toxicity. Despite significant commercial development, late stage clinical results of HBOC solutions hampered development. To evaluate the safety of PolyhHb as an O2 therapeutic agent in a vulnerable population, the present study was performed in a guinea pigs subjected to a high fat and high sucrose diet (HDHS). This model mimics human dyslipidemia and potentially induces endothelial dysfunction (ED). The objective of this study was to evaluate the impact of large molecular size PolyhHb on cardiovascular function in guinea pigs with dyslipidemia. Animals weighing between 150–200 g were fed a HDHS for 12 weeks. The metabolic characterization of the model included glucose tolerance testing (GTT) and measurement of the lipid profile. After metabolic characterization, the carotid artery and jugular vein were catheterized and catheters were exteriorized dorsally. Then, PolyhHb (n = 5) was subjected to a 20% blood volume (BV) exchange‐transfusion (BV estimated as 7.5% of body weight) with PolyhHb at 10 g/dL. Sham (n = 5) was subjected to the same procedure, but no exchange transfusion was performed. Post exchange, the animals were allowed to recover for 24 hours, after which blood pressure (BP) and heart rate (HR) were recorded, and BP and HR variability (HRV) were evaluated by spectral analysis. Animals were subjected to a sequence of vasoactive drugs to evaluate their baroreflex (phenylephrine, sodium nitroprusside, 1 and 2 ug), endothelial function (acethocoline (ACH), 2 and 4 ug), and nitric oxide response (Nitro‐L‐arginine methyl ester (L‐NAME), 12 mg/kg). Animals presented glucose intolerance and dyslipidemia. PolyhHb induced an 18% increase in MAP 24 hours after exchange‐transfusion. PolyhHb showed a decreased response to L‐NAME, suggesting a deficiency in nitric oxide (NO) dependent regulation of vascular tone, a known consequence of acellular Hb NO scavenging. There were no differences in baroreflex between groups nor significant endothelial dysfunction for the PolyhHb group compared to the Sham group. An increase in HRV and in sympathetic nervous system (SNS) modulation of the heart (low frequency band, LF) was observed in the PolyhHb group compared to Sham. On the other hand, parasympathetic modulation of the heart (high frequency band, HF) was decreased for the PolyhHb group compared to Sham, culminating in a 2‐fold increase in sympathetic vagal balance. Moreover, BP variability as well as blood vessel SNS modulation was increased for PolyhHb compared to Sham. In conclusion, PolyhHb transfusion impaired the autonomic nervous system in guinea pigs with dyslipidemia. Our results suggest that dyslipidemic patients might be more vulnerable to PolyhHb transfusion side effects.Support or Funding InformationThis work was supported by the NIH Heart Lung and Blood Institute under Grants T32‐HL105373, R01‐HL126945, and the DOD DMRDP under Grant W81XWH‐18‐1‐0059

BackgroundBlood transfusion as a treatment for anemia is vital in ensuring oxygen (O2) carrying capacity. However, fears of blood shortages and adverse reactions to stored blood transfusion have driven the development of oxygen carrying alternatives to blood. One class of alternatives to blood for transfusion medicine are hemoglobin (Hb) based oxygen carriers (HBOCs), which have been in development for decades. A recent advancement in the development of HBOCs is the discovery that their O2 affinity can be tightly regulated by locking Hb in the T state (low O2 affinity), or R state (high O2 affinity). By regulating O2 affinity, O2 extraction by tissues can be controlled, and targeted O2 delivery to hypoxic tissues is made possible. As the heart is one of the most sensitive organs to changes in oxygenation due to its high metabolic rate, the objective of this study was to test the efficacy of high and low O2 affinity HBOCs in maintaining cardiac function and systemic O2 delivery during severe anemia.MethodsPolymerized hemoglobin (PolyHb), the most scalable method of producing HBOCs, was synthesized in the high and low O2 affinity state (R and T, respectively) with glutaraldehyde and then subjected to 8–9 cycles of diafiltration as previously described. This resulted in a PolyHb solution containing only polymerized Hb molecules > 500kDa suspended in Lactated Ringers solution. Rats were instrumented with a pressure‐volume catheter to monitor cardiac function. Severe anemia was induced via a 50% isovolumic hemodilution with 5% human serum albumin. Rats were then resuscitated by a 40% isovolumic infusion of a 10 g/dL solution of R PolyHb, T PolyHb, or fresh blood (drawn from the animal during the first exchange).ResultsSevere anemia resulted in impaired cardiac function and O2 delivery. Transfusion of fresh blood, T PolyHb, and R PolyHb restored vascular resistance, blood pressure, and cardiac contractility. T PolyHb also promoted increased O2 extraction and myocardial energy utilization. Severe anemia did not cause significant changes in blood electrolytes relative to baseline. Severe anemia resulted in blood acidification that only recovered for animals resuscitated with T PolyHb. Furthermore, resuscitation with T PolyHb resulted in higher arterial pO2 and lower pCO2 than other groups. R PolyHb increased lactate and glucose, suggesting that the high O2 affinity prevented proper O2 release to some tissues, despite maintaining cardiac function.ConclusionsBoth T and R PolyHb maintain myocardial O2 extraction compared to fresh blood. T PolyHb appears to be the optimal molecule for increasing O2 delivery during anemic conditions. Although R PolyHb apparently decreased systemic O2 extraction during anemia, it can be potentially useful to increase O2 uptake in the lungs during hypoxic‐hypoxia. This study indicates that PolyHb is as efficacious as fresh blood in restoring cardiac function and oxygenation in rats during severe anemia.Support or Funding InformationThis work was supported by the NIH Heart Lung and Blood Institute under Grants T32‐HL105373, R01‐HL126945, and R01‐HL138116.