Oxygen Affinity Of Hemoglobin Research Articles

Rejuvenation solution as an adjunct cold storage solution maintains physiological haemoglobin oxygen affinity during early-storage period of red blood cells.

Red blood cell (RBC) units accumulate morphologic and metabolic lesions during storage before transfusion. Pyruvate-inosine-phosphate-adenine (PIPA) solutions (Rejuvesol, Biomet, Warsaw, IN) can be incubated with RBC units to mitigate storage lesions. This study proposes a PIPA treatment process, termed cold 'rejuvenation', using Rejuvesol as an adjunct additive solution, to prevent biomechanical storage lesions while avoiding the 1h PIPA incubation required with standard PIPA treatment. We compared the efficacy of cold to standard 'rejuvenation' in improving metabolic lesions that occur during cold storage of RBCs, without altering function. Twelve leucoreduced, A-positive RBC units were obtained. Each unit was aliquoted into either control (standard storage), washed (W), standard rejuvenation (SR) or cold rejuvenation (CR) groups, the latter two requiring washing. A volume-adjusted dose of Rejuvesol was instilled into the CR group upon receipt (Day 3). After 15days of storage, p50, RBC deformability, in-bag haemolysis and mechanical fragility were analysed. 'Any treatment' is defined as W, SR and CR, with comparisons in reference to control. Higher p50s were seen in rejuvenated groups (>30mmHg vs. <19mmHg; P<0·0001). Any treatment significantly increased elongation index (P=0·034) but did not significantly increase in-bag haemolysis (P=0·062). Mechanical fragility was not significantly different between groups (P=0·055) at baseline, but the control (CTL) group was more fragile after 2h in a cardiac bypass simulation than any treatment (P<0·0001). This study demonstrates that rejuvenation (standard or cold) prevents the leftward p50 shift of storage lesions without detrimental effect on RBC deformity, in-bag haemolysis or mechanical fragility.

Enhancement of erythrocyte oxygen transport function after hydrothermic storage under the action of human cord blood low-molecular fraction and the Actovegin drug

We study the effect of a low-molecular fraction (below 10 kDa) derived from human cord blood and Actovegin on the content of various forms of hemoglobin in erythrocytes stored under hypothermia (2-4 ºС) for 21 days. During the long-term storage of erythrocytes, there is known to be a change in the hemoglobin forms ratio toward the decrease of the relative content of oxyhemoglobin and an increase of deoxy- and methemoglobin. As a result, the affinity of hemoglobin for oxygen reduces. The incubation of erythrocytes in a rehabilitation medium with each of the studied low-molecular drugs was found to be crucial to increase the content of oxyhemoglobin and to reduce the amounts of deoxy- and methemoglobin relative to the control. As a result, there was a rise in the oxygenation coefficient, reflecting the oxygen saturation of hemoglobin, up to the level characteristic of freshly isolated erythrocytes. The data obtained show that the human cord blood low-molecular fraction and the Actovegin normalize the ratio of hemoglobin forms in hypothermically stored erythrocytes, but the use of the Actovegin drug is more effective.

Rising CO2 saves lives

Anthropogenic climate change is a multi-faceted problem. Rising CO2 levels in the atmosphere cause a warming of global temperatures, acidify the oceans and may ultimately deplete aquatic oxygen, leading to hypoxia. By itself, any one of these conditions can pose a real challenge to an animal, but how the stressors interact is still largely unexplored. Aquatic hypoxia may be especially intricate and, unlike the hypoxic conditions that one might experience when climbing a mountain, is almost always caused by organismal respiration in the water and, thus, is associated with an additional, simultaneous increase in CO2. The linkage between oxygen and CO2 levels in water has long been known, but how fish are affected by the combined challenge is poorly studied.To address the issue, Daniel Montgomery and his colleagues at the University of Exeter, UK, set out to test the effect of rising CO2 on the hypoxia tolerance of European seabass. They measured the oxygen consumption of the fish while lowering the available oxygen in the water; the point at which the animal can no longer extract oxygen from the depleted water and their oxygen consumption starts to decline is a popular indicator of hypoxia tolerance in fish. Knowing that higher levels of CO2 tend to acidify the blood of fish – compromising the ability of haemoglobin to bind oxygen and hindering the fish's ability to extract oxygen from the water – Montgomery then supplemented one of the treatments with a simultaneous increase in CO2, expecting that the combined challenge would render the fish more susceptible to hypoxia.However, contrary to these predictions, CO2-treated fish excelled at extracting oxygen from the water and outperformed the low-CO2 group in terms of hypoxia tolerance. When Montgomery took a closer look at the fish's blood, he found no difference in blood pH between the treatments, indicating a remarkable capacity of the seabass to counteract the acidifying effect of elevated CO2; surely this is good news for fish that must cope with global rises in CO2. In addition, fish in the rising CO2 treatment had a higher haemoglobin–oxygen affinity compared with the low-CO2 group – a surprising finding, given the common blood pH in the two groups – and this may explain their improved ability to tolerate hypoxia. Haemoglobin is housed within red blood cells, which may actively alter their internal environment to modulate oxygen transport. Whether this is part of the mechanism by which CO2-exposed seabass increased their haemoglobin–oxygen affinity remains to be seen.Studying the effects of climate change is complicated as numerous factors may interact to challenge an organism. Understandably, researchers often choose to study single factors in isolation, as this simplifies the interpretation of the data. However, Montgomery and colleagues show that overlooking interaction effects may limit our ability to predict the response of animals to climate change in the wild. In European seabass it appears that the combined condition of a decreasing oxygen level and an increasing CO2 level in the water, as is found in nature, improves hypoxia tolerance. To account for such effects in future work, current methods to assess hypoxia tolerance in fish should be revised to control for, or at least measure and report, CO2 levels in the water. Rising CO2 levels receive a lot of bad press and, in the context of climate change, rightfully so. However, at least for seabass that find themselves gasping for oxygen, the natural rise in CO2 that comes with aquatic hypoxia may actually save lives.

СОСТОЯНИЕ КИСЛОРОДТРАНСПОРТНОЙ ФУНКЦИИ КРОВИ ПРИ СИНДРОМЕ ОБСТРУКТИВНОГО АПНОЭ/ГИПОПНОЭ СНА

Summary. Оbstructive sleep apnea / hypopnea syndrome (OSAHS) is a topical medical problem and can lead to important medical consequences. OSAHS-associated biological changes are caused by periodic hypoxia and intermittent hypercapnia. The state and clinical significance of the oxygen transport function in OSAHS have not been studied and this was the purpose of the present research. The obtained results indicated the development of compensated metabolic acidosis during OSAHS: an increase in the lactate level with pH being within normal limits. There was a decrease in hemoglobin-oxygen affinity (a shift of oxyhemoglobin dissociation curve rightwards), regulating processes of tissues oxygenation during hypoxia. There also was a positive compensatory reaction in OSAHS. The identified changes correlated with the severity of sleep apnea.

Embryonic and Fetal Human Hemoglobins: Structures, Oxygen Binding, and Physiological Roles.

During the past two decades, significant advances have been made in our understanding of the human fetal and embryonic hemoglobins made possible by the availability of pure, highly characterized materials and novel methods, e.g., nano gel filtration, to study their properties and to correct some misconceptions. For example, whereas the structures of the human adult, fetal, and embryonic hemoglobins are very similar, it has generally been assumed that functional differences between them are due to primary sequence effects. However, more recent studies indicate that the strengths of the interactions between their subunits are very different leading to changes in their oxygen binding properties compared to adult hemoglobin. Fetal hemoglobin in the oxy conformation is a much stronger tetramer than adult hemoglobin and dissociates to dimers 70-times less than adult hemoglobin. This property may form the basis for its protective effect against malaria. A major source of the increased strength of fetal hemoglobin resides within the A-helix of its gamma subunit as demonstrated in studies with the hybrid hemoglobin Felix and related hybrids. Re-activating fetal hemoglobin synthesis in vivo is currently a major focus of clinical efforts designed to treat sickle cell anemia since it inhibits the aggregation of sickle hemoglobin. The mechanisms for both the increased oxygen affinity of fetal hemoglobin and its decreased response to DPG have been clarified. Acetylated fetal hemoglobin, which makes up 10-20% of total fetal hemoglobin, has a significantly weakened tetramer structure suggesting a similar role for other kinds of protein acetylation. Embryonic hemoglobins have the weakest tetramer and dimer structures. In general, the progressively increasing strength of the subunit interfaces of the hemoglobin family during development from the embryonic to the fetal and ultimately to the adult types correlates with their temporal appearance and disappearance in vivo, i.e., ontogeny.

Long-Term Hypoxic/Hypocapnic Storage of Red Blood Cells Results in Amelioration of Lesion Hallmarks and Increased In Vivorecovery at 24 Hours Post-Transfusion

Oxidative damage, dysmetabolism and decreased levels of 2, 3-diphosphoglycerate (2,3-DPG) affect red blood cell (RBC) survival and the affinity of Hemoglobin for oxygen are relevant hallmarks of RBC storage lesion, especially in specific clinical contexts. We hypothesized that oxygen and carbon dioxide reduction followed by hypoxic/hypocapnic storage would result in amelioration of RBC lesion assessed at the end of their shelf-life and at the shelf-life mode (21 days).To determine the relevance of hypoxic/hypocapnic storage in humans, a pivotal, prospective, randomized, two‐arm, crossover, three‐center trial was conducted to evaluate [Hemanext®] hypoxic/hypocapnic processing system and was used to assess whether this process and storage method met standard FDA acceptability criteria for long-term RBC storage. The Test arm consisted of whole blood (WB)‐derived, leukoreduced RBC in AS-3 additive processed at room temperature with the Hemanext system for 3 hours to achieve hypoxic/hypocapnic state within 12 hours of phlebotomy which was maintained hypoxic during storage for up to 43 days at 1‐6°C (Test).Unprocessed units (Control) were stored within 8 hours under conventional storage conditions. Subjects (N=100) donated CP2D WB (500 ± 50 mL) and a minimum of 93 pairs of RBC units per arm were analyzed for in vitroquality parameters. For in vivo analysis at end of storage, RBCs from 19 test subjects and 21 control subjects (14 paired) from two sites were radiolabeled with 51‐Cr/99‐Tc(m), autologously transfused, and analyzed for 24-hour recovery. Differences between the Test and Control groups were analyzed using the paired t-test (Wilcoxon test where necessary). Paired analyses of 24-hour in vivorecoveries on day 43 was 89.3±4.5% and 84.8±6.2% for the test and control, respectively; p&lt;0.01). Significantly higher levels of 2,3-DPG and adenosine 5'-triphosphate (ATP) were maintained for the Test group by days 21 and 42/43 of storage (Table 1). Percentage of hemolysis was similar in both groups. In summary, these data demonstrate that RBCs preserved in a user-friendly, self-contained hypoxic storage system are superior than the conventionally stored RBCs and may provide more viable RBCs for transfusion at 6 weeks of storage. Disclaimer: The51‐Cr/99‐Tc(m)labeling at one of the three sites occurred without prior RDRC approval and did not meet GMP standards. Disclosures Cancelas: Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Hemanext: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding; Velico: Consultancy, Research Funding. Yoshida:New Health Sciences Inc.: Employment, Equity Ownership. Dioguardi:Hemanext: Employment. Iselin:Hemanext: Employment. Dunham:Hemanext: Employment.

Differential Efficacy of Anti-Sickling and Anti-Inflammatory Mechanisms in a Fluorescent Intravital Microscopy Dorsal Skinfold Vaso-Occlusion Model in Sickle Cell Disease Townes Mice

Sickle cell disease (SCD) is characterized by acute and repetitive vaso-occlusive crises (VOC). These crises have been hypothesized to occur when blood flow is reduced following obstruction of sickle-shaped red blood cells in the vasculature. However, it is now well established that inflammation, oxidative stress, endothelial activation and pro-coagulation in sickle cell disease patients also contribute to the formation of heterocellular aggregates that can lead to VOC (Vercellotti and Belcher, 2014). Transgenic SCD mice recapitulate the pathology of human disease in response to stimuli such as heme injection and hypoxia/reoxygenation. SCD SS Townes mice, which express human α and sickle γAβS globins, AA Townes mice expressing normal human α and normal γAβA globins and heterozygous AS mice which express only one allele of the γAβS sickle gene were used. To characterize vaso-occlusion in these mice and evaluate the efficacy of different pharmacological mechanisms, we modified the skinfold vaso-occlusion model (Kalambur et al, 2004) using fluorescent intravital microscopy to visualize blood flow occlusion following hemin injection or hypoxia/reoxygenation challenge. Dorsal skinfold chambers were implanted and24 hours post-surgery mice were injected with FITC-dextran for visualization of flowing blood vessels. Skinfold bearing mice were then subjected to hemin treatment (16 μmoles/kg) or hypoxia (7%; 1 hour)/reoxygenation (1 hour) followed by the injection of Alexa fluor 647-labeled albumin to allow quantification of occluded vessels through dual fluorescent image analysis. Following hemin injection, SS mice showed significant ~30% vaso-occlusion in comparison to AA mice with ~8%, whereas the AS mice showed an intermediate phenotype with ~20% vaso-occlusion. Hypoxia/reoxygenation challenge also resulted in significant vaso-occlusion for SS mice (~25%) whereas only 5% was observed in AA mice. Interestingly, AS mice also showed a significant amount of vaso-occlusion (~25%) similar to SS mice when challenged with hypoxia/reoxygenation. Although no sickling can be observed in an ex vivo sickling assay using AS red blood cells, an intermediate amount of free Hemoglobin (Hb) can be detected in the plasma of these mice and rolling can be observed. This suggests that these vaso-occlusive models relate more on the inflammatory and endothelial activation state and are independent of the sickling potential of the red blood cell. We then used our model with hypoxia/reoxygenation challenge to evaluate the effects of dimethyl fumarate (DMF, 15 mpk BID), an anti-P-Selectin antibody (150ug/mouse) and the covalent hemoglobin oxygen affinity modulator GBT-440 (300 mpk). As anti-inflammatory agents, DMF and Anti-P-Selectin significantly reduced vaso-occlusion in SS mice by ~60% compared to the vehicle treated mice, but GBT-440 did not inhibit vaso-occlusion at a dose where a significant reduction in p50 was observed. In conclusion, our data have shown that obstruction of blood flow in the skinfold vaso-occlusion model in SCD Townes mice reflects the vascular inflammatory state of the disease and is independent of the ex vivo capacity of red blood cell to sickle. Disclosures Sturtevant: Sanofi: Employment. Macias-Garcia:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. van der Flier:Sanofi: Employment. Hicks:Sanofi: Employment. Demers:Sanofi: Employment.

Effects of Voxelotor (GBT440) Therapy on Laboratory Testing of Sickle Cell Patients

Background: Voxelotor (GBT440) is a small molecule allosteric effector with high specificity for hemoglobin (Hb) that inhibits Hb S polymerization by increasing hemoglobin oxygen affinity. Developed as an oral, once-daily medication, it is undergoing phase 3 randomized clinical trials in sickle cell disease patients. Therapeutic monitoring of Hb quantitative fractions is routine in sickle cell disease patients to assess medication and transfusion effects and future treatment requirements. In the clinical laboratory, some therapies can alter the Hb fraction percentages or characteristic protein migration/elution patterns and can mimic complex hemoglobinopathy disorders causing misinterpretation or unnecessary additional testing. We present the effects of voxelotor on hemoglobin identification/monitoring testing in multiple testing platforms. Methods: A whole blood anticoagulated sample from a male patient who was undergoing treatment for sickle cell disease was received for routine hemoglobin testing. Five additional samples with similar features were also received from three male patients (age range of all patients was 18 to 22 years) with sickle cell disease. Hemoglobin protein analysis was performed using standard methods including cation-exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE), isoelectric focusing (IEF), intact globin chain mass spectrometry (MS), sickle solubility and hemoglobin stability studies. DNA sequencing of the alpha (HBA1/HBA2) and beta globin (HBB) genes was performed on one sample. Results: CE (initial and 50:50 mix, figures 1A and 1B, respectively) revealed a Hb S peak with a shoulder and additional small peaks at position 203 (D zone) and 234 (cusp of zones E/A2). In some cases the 234 peak interfered with instrument designation of the A2 peak (50:50 mix with normal) and shifted the Hb S peak into the E zone which risks variant misidentification (figure 1C). Our laboratory overcame this limitation by substituting the normal sample with beta thalassemia trait to enhance the Hb A2 peak after mixing. HPLC tracings (figure 1D) showed a duplex peak pattern in the D and S windows with separate peak quantification percentages (index case showed elution at 4.18 and 4.37 minutes with 14.9% and 66.4%, respectively). The dual peaks detected by HPLC partially mimicked compound Hb S/D-Punjab. IEF (figure 1E) showed additional bands at -4.7, and -11.5 (Schneider-Barwick ratios). The additional peaks identified by CE and IEF mimicked an alpha chain hemoglobin variant; however, the expected hybrid patterns were atypical prompting further investigation. Sickle solubility was positive, heat stability studies were negative and mass spectrometry did not definitively detect abnormal peaks other than βS. DNA sequencing of the alpha and beta globin genes did not reveal abnormalities other than homozygous Hb S. Similar hemoglobin patterns were identified in all samples tested. Conclusion: Voxelotor treatment in sickle cell patients results in a distinctive pattern alteration in hemoglobin testing. Methods may not give accurate variant percentage estimation and the treatment may result in shifts in variant mobility causing erroneous laboratory results. Novel therapies are increasing in the treatment of hemoglobin disorders and medication effect should be a consideration when atypical patterns are encountered in the laboratory. Knowledge of these artifacts by clinicians and laboratorians is paramount to avoid erroneous diagnosis, unnecessary additional testing and inaccurate Hb variant quantitation for monitoring purposes. Notification of current treatment regimens to testing facilities is highly recommended in these patients. Figure 1 Disclosures No relevant conflicts of interest to declare.

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry.

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient's pO2 at which sickling starts and the minimal deformability of a patient's red blood cells, respectively. PoS is associated with an individual patient's hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Increased Hemoglobin Oxygen Affinity With 5-Hydroxymethylfurfural Supports Cardiac Function During Severe Hypoxia.

Acclimatization to hypoxia or high altitude involves physiological adaptation processes, to influence oxygen (O2) transport and utilization. Several natural products, including aromatic aldehydes and isothiocyanates stabilize the R-state of hemoglobin (Hb), increasing Hb-O2 affinity and Hb-O2 saturation. These products are a counter intuitive therapeutic strategy to increase O2 delivery during hypoxia. 5-Hydroxymethylfurfural (5-HMF) is well known Amadori compound formed during the Maillard reaction (the non-enzymatic browning and caramelization of carbohydrate-containing foods after thermal treatment), with well documented effects in Hb-O2 affinity. This study explores the therapeutic potential of 5-HMF on left ventricular (LV) cardiac function (LVCF) during hypoxia. Anesthetized Golden Syrian hamsters received 5-HMF i.v., at 100 mg/kg and were subjected to stepwise increased hypoxia (15, 10, and 5%) every 30 min. LVCF was assessed using a closed chest method with a miniaturized conductance catheter via continuous LV pressure-volume (PV) measurements. Heart hypoxic areas were studied using pimonidazole staining. 5-HMF improved cardiac indices, including stroke volume (SV), cardiac output (CO), ejection fraction (EF), and stroke work (SW) compared to the vehicle group. At 5% O2, SV, CO, EF, and SW were increased by 53, 42, 33, and 51% with 5-HMF relative to vehicle. Heart chronotropic activity was not statistically changed, suggesting that differences in LV-CF during hypoxia by 5-HMF were driven by volume dependent effects. Analysis of coronary blood flow and cardiac muscle metabolism suggest no direct pharmacological effects from 5-HMF, therefore these results can be attributed to 5-HMF-dependent increase in Hb-O2 affinity. These studies establish that naturally occurring aromatic aldehydes, such as 5-HMF, produce modification of hemoglobin oxygen affinity with promising therapeutic potential to increase O2 delivery during hypoxic hypoxia.

Modelling the relationships between haemoglobin oxygen affinity and the oxygen cascade in humans.

Haemoglobin affinity is an integral concept in exercise physiology that impacts oxygen uptake, delivery and consumption. How chronic alterations in haemoglobin affinity impact physiology is unknown. Using human haemoglobin variants, we demonstrate that the affinity of haemoglobin for oxygen is highly correlated with haemoglobin concentration. Using the Fick equation, we model how altered haemoglobin affinity and the associated haemoglobin concentration influences oxygen consumption at rest and during exercise via alterations in cardiac output and mixed-venous . The combination of low oxygen affinity haemoglobin and reduced haemoglobin concentration seen in vivo may be unable to support oxygen uptake during moderate or heavy exercise. The physiological implications, with regard to exercise, of altered haemoglobin affinity for oxygen are not fully understood. Data from the Mayo Clinic Laboratories database of rare human haemoglobin variants reveal a strong inverse correlation (r=-0.82) between blood haemoglobin concentration and P50 , an index of oxygen affinity [Hb=-0.3135(P50 )+23.636]. In the present study, observed P50 values for high, normal and low oxygen-affinity haemoglobin variants (13, 26 and 39mmHg) and corresponding haemoglobin concentrations (19.5, 15.5 and 11.4gdL-1 respectively) are used to model oxygen consumption as a fraction of delivery at rest ( =0.25Lmin-1 , cardiac output=5.70Lmin-1 ) and during exercise ( =2.75Lmin-1 , cardiac output=18.9lmin-1 ). With high-affinity haemoglobin, the model shows that normal levels of oxygen consumption can be achieved at rest and during exercise at the assumed cardiac output levels, with reduced oxygen extraction both at rest (16.8% high affinity vs. 21.7% normal) and during exercise (55.8% high affinity vs. 72.2% normal). With low-affinity haemoglobin, which predicts low haemoglobin concentration, oxygen consumption at rest can be sustained with the assumed cardiac output, with increased oxygen extraction (31.1% low affinity vs. 21.7% normal). However, exercise at 2.75lmin-1 cannot be achieved with the assumed cardiac output, even with 100% oxygen extraction. In conclusion, the model indicates chronic alterations in P50 associate directly with Hb concentration, highlighting that human Hb variants can serve as 'experiments of nature' to address fundamental hypotheses on oxygen transport and exercise.

New role for an old molecule: The 2,3-diphosphoglycerate case

BackgroundAerobic organisms have to overcame the dangerous species derived from the unquestionable favorable effects due to the utilization of oxygen in the cellular respiration. 2,3-Diphosphoglycerate (DPG) could be one of the molecules able to perform different role inside the cells and (from the data obtained from our experimental work) may help cellular components, in particular hemoglobin, to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS). MethodsTherefore, we have investigated the kinetic and antioxidant properties of this molecule against the main biological reactive species and the protective role of this molecules on hemoglobin treated with strong oxidant. ResultsDPG, at the physiological concentration is able to scavenge hydroxyl radical, peroxyl radical, cation radicals and to chelate iron in the reduced state. Moreover it is able to avoid oxidation of iron inside the hemoglobin following treatment with nitrite and tert-butyl hydroperoxide (t-BOOH). On the other side, it is not able to protect membrane components from oxidative burning. This different behavior towards radical species is probably linked to the polarity of the molecule and also the high levels of charged groups present on the surface of DPG, that avoid the possibility to act in an environment almost completely hydrophobic, as inside the membrane, where reactive species produce the main damages during the reactions of peroxidation. ConclusionsThis is the first paper dealing with the potential role of DPG not only as a modulator of oxygen affinity in hemoglobin, but also as a scavenger of radicals.

PF740 CEREBRAL BLOOD FLOW IN ADOLESCENTS WITH SICKLE CELL ANEMIA RECEIVING VOXELOTOR

Background:Oxygenated sickle hemoglobin (HbS) does not polymerize; therefore, increasing oxygen affinity to HbS is a therapeutic strategy for sickle cell anemia (SCA; HbSS and HbSβ0thalassemia), and voxelotor (GBT440) is a first‐in‐class, small molecule that increases the affinity of hemoglobin for oxygen. It has been postulated that increasing hemoglobin‐oxygen affinity could limit oxygen offloading from hemoglobin, thus impairing tissue oxygenation which could lead to an increase in cerebral blood flow and potential risk of stroke. This study used MRI techniques to evaluate cerebrovascular hemodynamics to inform on cerebral oxygenation with voxelotor therapy.Aims:To assess effects of voxelotor on grey matter cerebral blood flow (CBF) in children with SCA.Methods:This was a St. Jude Children's Research Hospital (St. Jude) investigator‐initiated, ancillary study of children enrolled in the HOPE‐KIDS1 (NCT02850406) trial. Study activities were approved by the St. Jude IRB and all participants provided documentation of informed consent. Participants underwent two non‐sedated functional magnetic resonance imaging with angiography (MRI/MRA) evaluations, once prior to receiving voxelotor and again following multiple doses (Table). Time resolved phase‐contrast (PC) MR imaging was used to measure CBF during multiple phases of the cardiac cycle at several anatomic locations, including the internal carotid arteries and anterior, middle and posterior cerebral arteries, and arterial spin labeling (ASL) sequences utilizing a multi‐channel head coil in a 3T scanner were performed.Results:Preliminary data on three patients are available. All three participants (Table) were being treated with a stable dose of hydroxyurea and had normal transcranial doppler velocities upon study entry. As per the HOPE‐KIDS1 study design, two received voxelotor at 900 mg per day and one received 1500 mg per day. Baseline MRI/MRA exams were normal in two participants and one had silent cerebral infarcts without vasculopathy. Generally, participants had higher hemoglobin levels and decreased hemolysis while receiving voxelotor. Two patients had lower global CBF while receiving voxelotor and the third showed essentially no change in CBF. Repeat MRI/MRAs showed no new or evolving areas of ischemia and EPO levels did not increase over time.Summary/Conclusion:Vasodilatory autoregulation of the cerebral vasculature enables a rapid and effective mechanism to titrate blood flow based on metabolic demand; increasing cerebral blood flow to offset impaired tissue oxygenation (Guilliams, K. Stroke 2019). In our small cohort of adolescents with SCA, the lack of increase in CBF after treatment with voxelotor suggests that there is no impairment in oxygen unloading to brain tissue. In fact, these preliminary findings of decreasing CBF with rising hemoglobin levels in two patients suggest improved oxygen delivery to the brain. Further study on the effects of voxelotor on cerebral hemodynamics and oxygen delivery is warranted.image

Physicochemical properties and oxygen affinity of glutaraldehyde polymerized crocodile hemoglobin: the new alternative hemoglobin source for hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers (HBOCs) are modified stroma-free hemoglobin molecules used in developing a blood substitute for therapeutic usage. In order to prevent hemoglobin dissociation, glutaraldehyde (GTA) was used to generate high-molecular weight heterogeneous crocodile hemoglobin (Poly-cHb). This work, Poly-cHb was created using various GTA concentrations, ranging from 0.025-0.150% (v/v). Physicochemical properties were investigated that were comparable GTA polymerized human hemoglobin (Poly-hHb). This study has revealed that GTA polymerization increases the molecular size of Native-cHbs from 14.10 nm over a range from 16.31 to 54.27 nm. Moreover, this polymerization alters the secondary structure and heme environment by decreasing the helicity ratio from 1.00 to 0.95 at the highest condition and exhibits hypochromic shift of the Soret band to be 0.88 times lower than the native. However, all Poly-cHbs still possessed higher oxygen affinity than that of Poly-hHbs with average P50 values of 13 and 21 mmHg, respectively. Although, polymerization affected the overall Poly-cHb structure slightly, but compensated by decreasing the denaturation level to lower than 10%. Thus, it is interesting to note that Poly-cHb may advantageously provide effective oxygen carriage and ability for pasteurization, which may benefit the search for new alternative hemoglobin sources for HBOC development.

Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients.

In sickle cell disease (SCD), sickle hemoglobin (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced deformability, leading to vaso‐occlusive crises and chronic hemolytic anemia. To date, there are no reliable laboratory parameters or assays capable of predicting disease severity or monitoring treatment effects. We here report on the oxygenscan, a newly developed method to measure RBC deformability (expressed as Elongation Index ‐ EI) as a function of pO2. Upon a standardized, 22 minute, automated cycle of deoxygenation (pO2 median 16 mmHg ± 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation <5%). In particular, physiological modulators of oxygen affinity, such as, pH and 2,3‐diphosphoglycerate showed a significant correlation (respectively R = ‑0.993 and R = 0.980) with Point of Sickling (PoS5%), which is defined as the pO2 where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with antisickling agents, including GBT440, which alter the oxygen affinity of hemoglobin, caused a reproducible left‐shift of the PoS, indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD patients were analyzed, we observed a significantly higher PoS in untreated homozygous SCD patients compared to treated patients and other genotypes. We conclude that the oxygenscan is a state‐of‐the‐art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of new treatment strategies and could have potential in prediction of complications.

Three classical papers in respiratory physiology by Christian Bohr (1855-1911) whose work is frequently cited but seldom read.

History has been kind to Christian Bohr (1855-1911). His name is attached eponymously to three different areas of respiratory physiology. The first is the Bohr dead space, which refers to the portion of the tidal volume that does not undergo gas exchange. The second is the increase in oxygen affinity of hemoglobin caused by the addition of carbon dioxide to the blood. This is known as the Bohr effect and is a very important feature of gas exchange, both in the lung and in peripheral tissues. Both of these contributions by Bohr are familiar to most students. Bohr's third contribution refers to the calculation of the changes in the Po2 of blood as oxygen is loaded in the pulmonary capillary, the so-called Bohr integration. This contribution is less well known now, partly because of the advent of digital computing, but it was important in its day. The analysis is challenging because the very nonlinear shape of the oxygen dissociation curve means that the Po2 difference between the alveolar gas and the capillary blood, which is the driving pressure for diffusion, changes in a complicated way. All three papers are in German, and two of them are long and tedious to read. English translations are available, but few people read the papers, despite the fact that the first two articles are very frequently cited. In the present article, Bohr's contributions are reviewed, and some parts of the articles that are particularly difficult to understand are clarified.

Association of endothelial nitric oxide synthase gene G894T polymorphism with blood oxygen transport

BackgroundNitric oxide (NO) is involved in formation of oxygen transport function of blood and oxygen transport in tissues by means of interaction with haemoglobin. Endothelial nitric oxide synthase gene G894T polymorphism affects the expression and the activity of the NO synthase enzyme. The influence of this polymorphism on the formation of oxygen-dependent processes in the body has not been completely investigated. ObjectiveTo evaluate the associations between G894T polymorphism of the endothelial nitric oxide synthase gene and the condition of oxygen transport function of blood in healthy male volunteers. MethodsThe study subjects were healthy young males aged 18–24 years (n = 165). The blood was drawn from the cubital vein at rest after 12 h following the last food intake. G984T polymorphism and the blood oxygen indices pO2, CvO2, SO2, pH, р50stand (the temperature was 37°С, рН = 7.4, рСО2 = 40 mm Hg) and р50act (actual temperature, рН and рСО2), etc. were determined. ResultsIn persons with the TT-genotype, the oxygen content in venous blood was 48.7% (q = 0.019) lower compared to subjects with the GT-genotype and 49.4% (q = 0.019) lower compared to subjects with the GG-genotype. The saturation of blood in carriers of the TT-genotype was 32.4% (q = 0.021) and 35.9% (q = 0.019) lower as opposed to subjects with the GT-genotype and the GG-genotype, respectively. In blood of subjects with the TT-genotype, the oxygen tension was 26.1% (q = 0.019) lower as compared to subjects with the GT-genotype and 27.4% (q = 0.019) lower as opposed to the GG-genotype. In turn, volunteers having a common allele in their genotype (GG + GT) demonstrated oxygen tension to be 26.7% (q = 0.019) higher compared to subjects with the TT-genotype. The blood pH values of the subjects having the recessive genotype were 0.022 units lower compared to the GG (q = 0.044) and GT (q = 0.042) genotypes. In volunteers with the TT-genotype, the p50stand parameter was 5.8% (q = 0.027) lower compared to subjects with the GT-genotype and 6.8% (q = 0.019) lower compared to volunteers with the GG-genotype. In persons with two T-alleles in their genotype, p50act was 5.4% (q = 0.019) lower compared to subjects with the GT-genotype and 6.4% (q = 0.019) lower compared to persons with the GG-genotype. ConclusionThe T-allele of G894T polymorphism is associated with low values for oxygen content, oxygen tension, acidic pH shift and increased haemoglobin affinity for oxygen under standard and real conditions. The presence of a minor allele in G894T polymorphism of the NOS3 gene contributes to formation of oxygen transport function of blood.

Structural modification of azolylacryloyl derivatives yields a novel class of covalent modifiers of hemoglobin as potential antisickling agents.

The intracellular polymerization and the concomitant sickling processes, central to the pathology of sickle cell disease, can be mitigated by increasing the oxygen affinity of sickle hemoglobin (HbS). Attempts to develop azolylacryloyl derivatives to covalently interact with βCys93 and destabilize the low-O2-affinity T-state (deoxygenated) HbS to the polymer resistant high-O2-affinity R-state (liganded) HbS were only partially successful. This was likely due to the azolylacryloyls carboxylate moiety directing the compounds to also bind in the central water cavity of deoxygenated Hb and stabilizing the T-state. We now report a second generation of KAUS compounds (KAUS-28, KAUS-33, KAUS-38, and KAUS-39) without the carboxylate moiety designed to bind exclusively to βCys93. As expected, the compounds showed reactivity with both free amino acid l-Cys and the Hb βCys93. At 2 mM concentrations, the compounds demonstrated increased Hb affinity for oxygen (6% to 15%) in vitro, while the previously reported imidazolylacryloyl carboxylate derivative, KAUS-15 only showed 4.5% increase. The increased O2 affinity effects were sustained through the experimental period of 12 h for KAUS-28, KAUS-33, and KAUS-38, suggesting conserved pharmacokinetic profiles. When incubated at 2 mM with red blood cells from patients with homozygous SS, the compounds inhibited erythrocyte sickling by 5% to 9%, respectively in correlation with the increase Hb-O2 affinity. These values compare to 2% for KAUS-15. When tested with healthy mice, KAUS-38 showed very low toxicity.

Tibetan Enriched PKLR Variant Is Beneficial to High Altitude Adaption By Improving Oxygen Delivery

Tibetans have been living at altitudes over 3500 m for ~20,000 years and have developed unique beneficial evolutionary genetic adaptions (PMID:28448578). Our previous study identified selected genetic haplotypes in two genes, EPAS1 (encoding hypoxia-inducible factor 2-alpha [HIF2-a], a transcription factor that mediates the hypoxic response), and EGLN1 (encoding prolyl hydroxylase 2 [PHD2], a principal negative regulator of HIF stability (PMID:25129147). The presence of these two haplotypes correlates with lower hemoglobin levels in Tibetans compared to other highlanders. However, the entire diverse complex of molecular mechanisms of high altitude adaptation is still largely unknown and our study showed that neither EPAS1 nor EGLN1 variants fully explain the mechanism of protection from polycythemia in Tibetans in high altitude (PMID:28233034).We found an enriched haplotype in the PKLR gene (encoding pyruvate kinase [PK] expressed only in liver and red blood cells). The PK enzyme is in the terminal portion of the glycolytic pathway, and its decreased activity leads to accumulation of proximal glycolytic intermediates, including 2,3-diphosphoglycerate (DPG) which shifts the hemoglobin dissociation curve to right (high p50) and increases oxygen release to tissues from a unit of hemoglobin. We hypothesized that Tibetan enriched PKLR variants might improve oxygen delivery to tissues and help explain the protection from polycythemia at high altitude.Genomic analyses revealed that this PKLR haplotype is enriched in Tibetans but is not unique to Tibetans. It has the highest frequency in Tibetans (89%), with a lower prevalence in Chinese and Mongolians (~77%), Kyrgyz (~60 %), Aymara (~44 %), and Colombians (~30 %) and a much lower frequency in Caucasians (11%), perhaps explaining the heterogeneity of responses to hypoxia within and among these populations. Our study of reticulocyte RNA showed that transcript levels of PKLR progressively decrease with increasing altitude in controls and even more in Tibetans with the Tibetan evolutionary selected PKLR haplotype. Tibetans with the PKLR haplotype (heterozygotes and homozygotes) have lower PKLR transcript levels than wild type Tibetans. Because of the paucity of wildtype PKLR haplotype in Tibetans and the challenges of acquiring Tibetan samples in high altitude in China, we collected samples from 125 m (Cheboksary, Chuvashia); 800 m (Bishkek, Kyrgyzstan) and 2640 m (Bogota, Colombia). PK activity, PKLR transcript levels, and ATP decreased at 2640 m compared to 800 m, while p50 increased at 2640 m. PKLR transcript levels correlated with PK activity and ATP and inversely correlated with p50. PK activities also correlated with PKLR transcript levels and ATP and inversely correlated with p50. At 2640 m, PK activity was lower and p50 levels were higher in those with the enriched PKLR haplotype. These results demonstrate that increasing altitude decreases PK activity, resulting in increasing p50 providing a molecular basis for the previously reported improvement of oxygen delivery at high altitude (PMID:17394415).To study the roles of HIFs in the regulation of PKLR gene expression, we also collected samples from Chuvash polycythemia (CP) homozygotes and Chuvash controls. CP homozygotes have a mutation in the VHL gene, a negative regulator of HIFs, that results in augmented HIF levels. CP homozygotes had lower PKLR mRNA in reticulocytes, PK activity, and PKR protein levels in red blood cells compared to controls, while their 2,3 DPG levels were higher. These data confirm that PKLR expression levels are negatively regulated by HIFs.Our findings demonstrate that individuals in high altitudes have lower PKLR transcript levels and PK activity, resulting in high 2,3DPG and p50 which shifts the hemoglobin dissociation curve to right. This decreases affinity of hemoglobin for oxygen, which improves tissue oxygen delivery and as such is another mechanism in Tibetans that protects from high altitude polycythemia. We also demonstrate that HIFs negatively regulate PKLR expression, leading to better oxygen release from hemoglobin at high altitude. DisclosuresKosinski:Agios: Employment, Equity Ownership. Kung:Agios: Employment, Equity Ownership. van Wijk:RR Mechatronics: Research Funding; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

LC-MS Analysis of Anti-Sickling Compounds in Cord Blood Derived RBCs Demonstrates Modification of Fetal Hemoglobin and Globin Chain Binding Preferences

Sickle cell disease (SCD) is a genetic hemoglobinopathy driven largely by a single codon mutation of the β-globin gene resulting in polymerization of hemoglobin S (HbS). Anti-sickling approaches that involve increasing the oxygen affinity of HbS to treat SCD are under development and offer the potential to directly prevent HbS polymerization and its downstream pathophysiology. Two such compounds, 5-hydroxymethylfurfural (5HMF) and voxelotor (GBT440) have entered clinical trials for SCD with promising results and exert their therapeutic effects by modifying the N-terminus of HbS α-globin chains to form a reversible Schiff base. Formation of this N-terminal adduct stabilizes the oxygen-bound R-state (in the R2 conformation) that increases the oxygen affinity of the altered HbS and delaying the polymerization of HbS. In addition, genetic and small molecule therapies designed to increase fetal hemoglobin (HbF) expression hold great potential for the treatment of SCD. Increasing the percentage of HbF in RBCs significantly slows sickling kinetics without affecting oxygen delivery. Combination approaches of high-O2-Hb modification with HbF inducing therapies clinically could result in increased efficacy in the treatment of SCD, but the impact of hemoglobin modifiers on fetal hemoglobin has not been reported. Our present studies investigated the effects of 5HMF and voxelotor in HbF-rich umbilical cord blood derived RBCs.HbF-rich (60-90%) RBCs were isolated from cord blood and incubated with commercially available 5HMF and voxelotor synthesized in-house. The effect of these compounds on hemoglobin oxygen affinity was determined by measuring the p50 of the oxygen saturation curve in whole cells. Sites of modification were determined directly by incubating compounds with the purified RBC lysate, stabilizing the N-terminal adduct by reduction to the amine, and analysis of the resulting modification by LC-MS. Similar to the reported p50 shifts with normal adult hemoglobin (HbA) and HbS, 5HMF and voxelotor increased the oxygen-binding affinity of HbF with an EC50 of 7.9 mM and 560 mM respectively. 1 mM voxelotor lowered cord RBC p50 to 4 mmHg in vitro. LC-MS analysis showed that 5HMF exclusively modified the N-terminus of the α-globin chain, with no modification of b-globin and g-globin chains. Unexpectedly, the α-globin, β-globin and γ-globin chains were all modified by voxelotor following incubation with cord blood. Voxelotor was also shown to modify both α-globin and the β-globin or βS-globin chains on purified HbA or HbS, respectively. These data contrast with published crystallography data demonstrating that voxelotor selectively modifies a single α-globin chain in CO-ligated HbS (Oksenberg et al 2016).Although anti-sickling aromatic aldehydes have similar effects on the oxygen binding affinity of HbA, HbS and HbF, they can vary in their selectivity for modification of the α-globin and beta-like chains of HbF, HbA, and HbS (Abraham et al. 1995). To further investigate our data with voxelotor and increase our understanding of this class of molecules, other hemoglobin modifying aldehyde molecules such as 5-formylsalicyclic acid (5FSA), tucaresol and velaresol (BW12C) will be examined. DisclosuresVieira:Bioverativ a Sanofi Company: Employment. Hong:Bioverativ a Sanofi Company: Employment, Equity Ownership. Desai:Bioverativ a Sanofi Company: Employment, Equity Ownership. Safo:Bioverativ a Sanofi Company: Consultancy; Virginia Commonwealth University: Employment. Light:Bioverativ a Sanofi Company: Employment, Equity Ownership.