Increase In Oxygen Affinity Of Hemoglobin Research Articles

Effects of Carbon Dioxide and Temperature on the Oxygen-Hemoglobin Dissociation Curve of Human Blood: Implications for Avalanche Victims.

Completely avalanche-buried patients are frequently exposed to a combination of hypoxia and hypercapnia with a risk of normothermic cardiac arrest. Patients with a long burial time and an air pocket are exposed to a combination of hypoxia, hypercapnia, and hypothermia which may lead to the development of the “triple H syndrome”. This specific combination has several pathophysiological implications, particularly on the cardiovascular system and oxygen transport (oxygen supply and oxygen consumption). To examine the effects on hemoglobin oxygen affinity, we investigated venous blood samples from 15 female and 15 male healthy subjects. In a factorial design of four different carbon dioxide partial pressure (PCO2) levels (20, 40, 60, and 80 mmHg) and five different temperature levels (13.7°C, 23°C, 30°C, 37°C, and 42°C), 30 unbuffered whole blood samples were analyzed in a newly developed in vitro method for high-throughput oxygen dissociation curve (ODC) measurements. P50s, Hill coefficients, CO2-Bohr coefficients, and temperature coefficients were analyzed using a linear mixed model (LMM). Mean P50 at baseline (37°C, 40 mmHg PCO2) was 27.1 ± 2.6 mmHg. Both CO2-Bohr (p < 0.001) and temperature coefficients (p < 0.001) had a significant effect on P50. The absolute CO2 effect was still pronounced at normothermic and febrile temperatures, whereas at low temperatures, the relative CO2 effect (expressed by CO2-Bohr coefficient; p < 0.001, interaction) was increased. The larger impact of PCO2 on oxygen affinity at low temperature may be caused by the competition of 2,3-BPG with PCO2 and the exothermic binding characteristic of 2,3-BPG. In a model of an avalanche burial, based on published data of CO2 levels and cooling rates, we calculated the resulting P50 for this specific condition based on the here-reported PCO2 and temperature effect on ODC. Depending on the degree of hypercapnia and hypothermia, a potentially beneficial increase in hemoglobin oxygen affinity in the hypoxic condition might ensue.

Estimation of Achievable Oxygen Consumption Following Transfusion With Rejuvenated Red Blood Cells

Erythrocyte storage induces a nonphysiological increase in hemoglobin-oxygen affinity (quantified by low p50, the oxygen tension at 50% hemoglobin saturation), which can be restored through biochemical rejuvenation. The objective was to mathematically model the impact of transfusing up to 3 standard allogeneic units or rejuvenated units on oxygen delivery (DO2) and oxygen consumption (VO2). Oxygen dissociation curves were generated from additive solution-1 red blood cell (RBC) leukoreduced units (n = 7) before and after rejuvenation following manufacturer's instructions. Two of these units were used to prepare standard or rejuvenated donor RBC and added to samples of fresh whole blood. These admixtures were used to construct an in vitro transfusion model of postoperative anemia and determine a linear equation for calculating the sample p50, which was subsequently used to calculate DO2 and VO2 after simulated transfusions. Whole blood-packed red blood cell unit admixture p50s could be predicted from a linear model including the p50 of its components, the mass fraction of the transfused component, and interaction terms (R2 = .99, P < 0.001). Transfusion with standard units slightly, but significantly, increased projected DO2 compared with rejuvenated units (P = 0.03), but rejuvenated units markedly increased projected VO2 (P = 0.03). Standard units did not significantly change VO2 relative to pre-transfusion levels (P > 0.1). Using high-p50, rejuvenated RBC in simulated transfusions greatly improved projected VO2, indicating the potential for increased end-organ oxygen availability compared with standard transfusion. Patient capacity to increase cardiac output after cardiac surgery may be limited. Transfusing high-p50 RBC in this setting may improve the perioperative care of these patients.

Allosteric mechanisms underlying the adaptive increase in hemoglobin-oxygen affinity of the bar-headed goose.

The high blood-O2 affinity of the bar-headed goose (Anser indicus) is an integral component of the biochemical and physiological adaptations that allow this hypoxia-tolerant species to undertake migratory flights over the Himalayas. The high blood-O2 affinity of this species was originally attributed to a single amino acid substitution of the major hemoglobin (Hb) isoform, HbA, which was thought to destabilize the low-affinity T state, thereby shifting the T-R allosteric equilibrium towards the high-affinity R state. Surprisingly, this mechanistic hypothesis has never been addressed using native proteins purified from blood. Here, we report a detailed analysis of O2 equilibria and kinetics of native major HbA and minor HbD isoforms from bar-headed goose and greylag goose (Anser anser), a strictly lowland species, to identify and characterize the mechanistic basis for the adaptive change in Hb function. We find that HbA and HbD of bar-headed goose have consistently higher O2 affinities than those of the greylag goose. The corresponding Hb isoforms of the two species are equally responsive to physiological allosteric cofactors and have similar Bohr effects. Thermodynamic analyses of O2 equilibrium curves according to the two-state Monod-Wyman-Changeaux model revealed higher R-state O2 affinities in the bar-headed goose Hbs, associated with lower O2 dissociation rates, compared with the greylag goose. Conversely, the T state was not destabilized and the T-R allosteric equilibrium was unaltered in bar-headed goose Hbs. The physiological implication of these results is that increased R-state affinity allows for enhanced O2 saturation in the lungs during hypoxia, but without impairing O2 delivery to tissues.

Alteration of the α1β2/α2β1 subunit interface contributes to the increased hemoglobin-oxygen affinity of high-altitude deer mice.

BackgroundDeer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant.ResultsHighland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules.ConclusionsThe analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1β2/α2β1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.

Increased hemoglobin-oxygen affinity ameliorates bleomycin-induced hypoxemia and pulmonary fibrosis.

Although exertional dyspnea and worsening hypoxia are hallmark clinical features of idiopathic pulmonary fibrosis (IPF), no drug currently available could treat them. GBT1118 is a novel orally bioavailable small molecule that binds to hemoglobin and produces a concentration‐dependent left shift of the oxygen–hemoglobin dissociation curve with subsequent increase in hemoglobin–oxygen affinity and arterial oxygen loading. To assess whether pharmacological modification of hemoglobin–oxygen affinity could ameliorate hypoxemia associated with lung fibrosis, we evaluated GBT1118 in a bleomycin‐induced mouse model of hypoxemia and fibrosis. After pulmonary fibrosis and hypoxemia were induced, GBT1118 was administered for eight consecutive days. Hypoxemia was determined by monitoring arterial oxygen saturation, while the severity of pulmonary fibrosis was assessed by histopathological evaluation and determination of collagen and leukocyte levels in bronchoalveolar lavage fluid. We found that hemoglobin modification by GBT1118 had strong antihypoxemic therapeutic effects with improved arterial oxygen saturation to near normal level. Moreover, GBT1118 treatment significantly attenuated bleomycin‐induced lung fibrosis, collagen accumulation, body weight loss, and leukocyte infiltration. This study is the first to suggest the beneficial effects of hemoglobin modification in fibrotic lungs and offers a promising and novel therapeutic strategy for the treatment of hypoxemia associated with chronic fibrotic lung disorders in human, including IPF.

GBT440, a Potent Anti-Sickling Hemoglobin Modifier Reduces Hemolysis, Improves Anemia and Nearly Eliminates Sickle Cells in Peripheral Blood of Patients with Sickle Cell Disease

▪Background: Sickle cell disease (SCD) is caused by polymerization of Hemoglobin S, resulting in red blood cell (RBC) sickling, RBC destruction, vaso-occlusive episodes and end-organ damage. No direct anti-polymerization, mechanism-based, preventive therapy is available. GBT440 is a novel small molecule hemoglobin modifier which increases hemoglobin oxygen affinity. In vitro and in vivo studies have shown it to be a potent and direct anti-sickling agent with high specificity for hemoglobin, and that 10-30% hemoglobin modification could be both safe (not compromising oxygen delivery) and effective (preventing HbS polymerization). We hypothesized that a potent antisickling hemoglobin modifier should rapidly interrupt RBC hemolysis, improve anemia and potentially become a safe and effective long-term disease-modifying therapy. We therefore explored safety, pharmacokinetics and pharmacodynamics and potential efficacy in healthy volunteers and SCD patients.Methods: This prospective, randomized, placebo-controlled, double blind, parallel group phase I/II study enrolled healthy volunteers (HV) ages 18-55, and homozygous HbSS SCD patients ages 18-60 with baseline Hb levels ³6 g/dL and ²10 g/dL and without vaso-occlusive crisis or transfusion within 30 days of screening. The study was conducted in two parts: part A tested single ascending doses and part B multiple ascending doses of study drug with 6:2 randomization (GBT440:placebo). Doses administered were: part A, HV cohorts 100mg to 2800 mg, SCD cohort 1000 mg; part B, HV cohorts 300 mg to 900 mg once daily for 15 days, SCD cohort 700 mg once daily for 28 days. The primary endpoint was safety. Secondary endpoints included pharmacokinetics (PK) and pharmacodynamics (PD). Clinical indices of hemolysis were prespecified as endpoints and are described for the SCD multiple dose cohort (700 mg QD for 28 days). Descriptive statistics were used to analyze data.Results: As of July 24 2015, 64 healthy volunteers (HVs) and 16 SCD patients had been enrolled. 54 healthy volunteers had completed the study, 2 were discontinued due to mild-moderate nonserious adverse events (headache, rash) and 8 were in follow-up; 8 SCD patients had completed Part A of the study and 8 were in Part B follow-up. No SCD patients were discontinued; one part B subject had a dose reduction from 700 mg to 400 mg (due to abdominal discomfort). GBT440 was generally well-tolerated, most adverse events were mild, there were no deaths, and there was one serious adverse event (AE) of acute painful crisis in a placebo subject. There were no AEs related to tissue hypoxia. GBT440 showed dose proportional PK, a terminal half-life of 1.5-3 days, high partitioning into RBCs (RBC:Plasma 60-90:1), and a dose dependent increase in hemoglobin oxygen affinity in both HV and SCD patients. In the multiple dose SCD cohort, all patients were evaluable at 28 days (age range 21-52 years; 2 were on hydroxyurea; 3 males/5 females). GBT440-treated patients showed increased hemoglobin, hematocrit and erythrocyte counts with corresponding decreases in LDH, unconjugated bilirubin, reticulocytes and erythropoietin levels with trends evident as early as day 8 compared to no changes in the placebo group (Figure 1 and Table 1). Analysis of peripheral blood smears revealed a marked reduction in sickle cells with GBT440 treatment.Conclusions: Single, oral, daily dosing with GBT440 was well tolerated across a wide dose range and demonstrated dose proportional and predictable PK and PD. GBT440 demonstrated proof of mechanism with a dose-dependent increase in hemoglobin oxygen affinity without causing tissue hypoxia. GBT440 demonstrated proof of concept in SCD patients with rapid reduction in RBC hemolysis and improved oxygen delivery to tissues as evidenced by reduced erythropoietin level, and a marked reduction in circulating sickle cells. These results support further clinical investigation of GBT440 as a potential disease-modifying therapy for SCD.Table 1Mean change to Day 28 (SEM)GBT440PLAHemoglogin (g/dL)0.8 (0.2)-0.5 (0.4)% Sickle cells in peripheral blood (%)-83 (9)19 (4)Erythrocyte count (1012/L)0.5 (0.1)-0.1 (0.2)Reticulocyte count (%)-2.2 (1)0.8 (1.6)LDH (% change from baseline)-12 (7)-9 (1)Unconjugated bilirubin (% change from baseline)-25 (10)-9.6 (16)Erythropoietin (U/L)-29 (16)21 (33)GBT = GBT440 treated subject; PLA = placebo subject; SEM = standard error of the mean [Display omitted] DisclosuresLehrer-Graiwer:Global Blood Therapeutics: Employment, Equity Ownership. Howard:Pfizer: Consultancy; Novartis: Consultancy, Other: Travel Grant; Aes-Rx: Consultancy. Layton:Agios: Consultancy; Novartis: Consultancy; Glaxo Smith Kline: Consultancy. Mant:Quintiles: Employment, Equity Ownership. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Koller:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Patel:Global Blood Therapeutics: Employment, Equity Ownership. Ramos:Global Blood Therapeutics: Employment, Equity Ownership.

Effect of emotional stress on hemoglobin oxygen affinity in low resistant animals under normal conditions and during cerebral ischemia

Hemoglobin oxygen affinity within the estimated physiological range plays an adaptive antioxidant role during acute cerebral ischemia. This range depends on individual emotional resistance. Brain ischemia induced by common carotid artery occlusion in low resistant Wistar rats increased hemoglobin oxygen affinity by 12% during the acute period. Emotional stress also increased hemoglobin oxygen affinity and determined shifts in this parameter during the development of cerebral ischemia: moderate increase in hemoglobin oxygen affinity (<25%) was followed by further increase in this parameter, while more pronounced shift (>25%) resulted in a significant drop in this parameter due to hemoglobin deoxygenation. Adaptation to stress shifted the upper physiological limit for self-regulation of this process.

Factorial scopes of cardio-metabolic variables remain constant with changes in body temperature in the varanid lizard,Varanus rosenbergi

The majority of information concerning the cardio-metabolic performance of varanids during exercise is limited to a few species at their preferred body temperature (T(b)) even though, being ectotherms, varanids naturally experience rather large changes in T(b). Although it is well established that absolute aerobic scope declines with decreasing T(b), it is not known whether changes in cardiac output (V(b)) and/or tissue oxygen extraction, (Ca(O2) - Cv(O2)), are in proportion to the rate of oxygen consumption (Vo(2)). To test this, we studied six Rosenberg's goannas (Varanus rosenbergi) while at rest and while maximally exercising on a treadmill both at 25 and 36 degrees C. During maximum exercise both at 25 and 36 degrees C, mass-specific rate of oxygen consumption (Vo(2kg)) increased with an absolute scope of 8.5 ml min(-1) kg(-1) and 15.7 ml min(-1) kg(-1), respectively. Interestingly, the factorial aerobic scope was temperature-independent and remained at 7.0 which, at each T(b), was primarily the result of an increase in V(bkg), governed by approximate twofold increases both in heart rate (f(H)) and cardiac stroke volume (V(Skg)). Both at 25 degrees C and 36 degrees C, the increase in V(bkg) alone was not sufficient to provide all of the additional oxygen required to attain maximal Vo(2kg), as indicated by a decrease in the blood convection requirement V(bkg)/Vo(2kg); hence, there was a compensatory twofold increase in (Ca(O2) - Cv(O2)). Although associated with an increase in hemoglobin-oxygen affinity, a decrease in T(b) did not impair unloading of oxygen at the tissues and act to reduce (Ca(O2) - Cv(O2)); both Ca(O2)) and Cv(O2)) were maintained across T(b). The change in Vo(2kg) with T(b), therefore, is solely reliant on the thermal dependence of V(bkg). Maintaining a high factorial aerobic scope across a range of T(b) confers an advantage in that cooler animals can achieve higher absolute aerobic scopes and presumably improved aerobic performance than would otherwise be achievable.

Mechanism of the effect of alkalosis on maximum oxygen uptake in hypoxic exercise

Maximum oxygen uptake (V̇ O 2max ) of rats is increased in alkalosis; this increase is larger in hypoxic than in normoxic exercise (Gonzalez et al. (1991b). J. Appl. Physiol., 71: 1050–1056). The objective of the present experiments was to determine the mechanism of this phenomenon. NaHCO 3 (0.3 M, 9 mmol/kg) was given 15 min before exercise to rats acclimated to P b 380 Torr for 3 weeks (HxBic) and to normoxic rats (NxBic). Additional groups of acclimated and normoxic rats received 0.3 M NaCl (HxNaCl and NxNaCl, respectively). Hx rats exercised at P i O 2 ≈ 70 Torr ; Nx rats at P i O 2 ≈ 140 Torr . V̇ O 2max was higher after NaHCO 3 than after NaCl; this effect was larger in hypoxia. The increase in V̇ O 2max was mediated through an increased arterio-venous oxygen concentration difference ((Ca - Cv) O 2 ), due, in turn, to a higher Ca O 2 without changes in Cv O 2 . (Ca - Cv) O 2 after NaHCO 3 was larger in hypoxia because a change in pH results in a larger change in HbO 2 saturation at the values prevalent in arterial blood of the hypoxic rats ( i.e. P O 2 ∼ 40–50 Torr, S O 2 50–60%), than at either the values of normoxic arterial blood or of venous blood during maximum exercise. These results suggest that an increase in hemoglobin-oxygen affinity improves oxygen uptake in the lungs during hypoxia, without hindering its release in the tissues, and therefore has a beneficial effect in hypoxic exercise.

The Effect of Hemodialysis on Oxygen Transport in Chronic Uremics

Previous studies from our laboratory suggested a decrease in tissue oxygen delivered during hemodialysis of chronic uremic patients due to an increase in hemoglobin-oxygen affinity, i.e. decrease in P50. This current study was designed to determine whether the changes in cardiac index and/or tissue oxygen extraction could compensate for increases in hemoglobin-oxygen affinity previously observed, so that total tissue oxygen delivery was unchanged during hemodialysis. This study demonstrated in patients during a 6-hour hemodialysis that: 1) no change occurs in hemoglobin-oxygen affinity expressed as P50, with unchanging plasma inorganic phosphate and red blood cell pH; 2) increased extraction of oxygen from tissue, measured via (A-V) O2 difference compensates for decreased oxygen delivery due to decreased cardiac output; and although there was no demonstrable change in hemoglobin-oxygen affinity, it would appear that increased tissue extraction of oxygen via other mechanisms should be easily able to compensate for previously reported changes in P50.

Effect of potassium cyanate on carbon monoxide and hypoxic hypoxia‐induced lethality

Pretreatment of mice with potassium cyanate for 15 days resulted in a significant increase in hemoglobin-oxygen affinity (decrease in P 50) and a significant protection against hypoxic hypoxia-induced lethality. However, cyanate pretreatment had no significant effect on carbon monoxide-induced lethality. Oxygen consumption in cyanate-treated mice was significantly lower than in controls in room air but not different in hypoxic environments. Whether the protective effect of cyanate on hypoxic hypoxia-induced lethality is due to the increase in hemoglobin-oxygen affinity or attributable to a non-hemoglobin-related action of cyanate remains unresolved. Regardless, the need exists to reevaluate the current concept that changes in hemoglobin affinity for oxygen correlate with susceptibility to hypoxemic states. In addition, data presented together with previous findings support the concept that carbon monoxide lethality is not solely attributable to a carboxyhemoglobin-induced hypoxemia.

Zinc in the treatment of homozygous sickle cell anemia: studies in an animal model.

We have studied the effects of zinc on the 51Cr survival of red blood cells (RBC) from patients with homozygous sickle cell anemia (SCA) using an animal model in which the RBC were transfused into specially prepared rats. The slope (lambdas) of a standard 51Cr RBC survival curve was used as a measure of the rate of RBC sequestration. The effects of intravenous zinc were of considerable therapeutic interest from the standpoint of setting guidelines for effective blood levels of zinc in patients. SCA RBC were transfused into rats whose plasma zinc levels had been raised 3-6 times above normal (300-600 mug/100 ml) by prior iv injection of zinc acetate; in three experiments the mean lambdas in zinc-treated animals breathing 15-16% oxygen was significantly lower (meaning lessened sequestration and greater survival) than saline-treated controls. A possible explanation for the requirement to lower ambient oxygen tension in order to see this zinc effect is discussed. We have further observed an increased mean lambdas for RBC from 10 SCA patients compared to 4 normal controls (0.134 vs 0.030; t = 2.8, p less than 0.01). The lambdas values are quite patient specific (4 patients studied; F = 18.2, P = 0.002). In vitro pretransfusion treatment of SCA RBC with 1.5 mM zinc resulted in a significant increase in hemoglobin oxygen affinity and a marked reduction in lambdas (0.073 vs 0.120; t = 4.5, p less than 0.01). The mean lambdas was not affected by in vitro 0.3 mM zinc treatment; this level did not change hemoglobin oxygen affinity. We conclude that systemic zinc therapy in the animal model described, at plasma levels only slightly higher than those presently obtained in patients, prolongs SCA RBC survival. This animal model is a sensitive measure of the sicklability of SCA RBC and is useful in the testing of in vitro and in vivo antisickling agents.

Reduced red cell 2,3-diphosphoglycerate and adenosine triphosphate, hypophosphatemia, and increased hemoglobin-oxygen affinity after cardiac surgery.

Serum inorganic phosphorous was decreased significantly on the third postoperative day following cardiac surgery in 18 patients initially studied. Reduced plasma inorganic phosphate has been shown to cause a reduced concentration of red cell organic phosphates, an important determinant of hemoglobin-oxygen affinity. Therefore, 10 consecutive patients were studied to determine if reduced 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) concentration and increased hemoglobin-oxygen affinity accompanied the fall in serum inorganic phosphate concentration. A significant fall in 2,3-DPG and an increase in hemoglobin-oxygen affinity was present in red cells of patients studied on the first postoperative day. A reduction in red cell ATP was also present and persisted for 5 days during which time red cell 2,3-DPG returned to levels which were in excess of preoperative values. The reduction in serum inorganic phosphorous followed the reduction in red cell 2,3-DPG and correlated with the reduction in ATP. The latter changes may indicate the diversion of glucose and more specifically 1,3-DPG into the Rapoport-Leubering pathway away from ATP generation at the phosphoglycerate kinase step and the utilization of plasma inorganic phosphate for 2,3-DPG resynthesis. Neither the transfusion of stored blood nor the effect of cardiopulmonary bypass fully explained the reduction in red cell 2,3-DPG and the inefficiency of hemoglobin function postoperatively. Further studies in postsurgical patients are needed to clarify the cause of the changes observed since they are potentially deleterious, especially in the subject with compromised cardiovascular and pulmonary function.