Increased Oxygen Affinity Research Articles

Hb San Cataldo [β144(HC1)Lys→Thr; HBB: C.434A > C]: A New Hemoglobin Variant with Increased Affinity for Oxygen

A 59-year-old Italian woman came to our center for revaluation of a previous diagnosis of polycythemia vera. The patient presented with a lifelong history of polycythemia, no increase in white blood cells (WBCs) and platelets, and a negative bone marrow biopsy. Analysis of hemoglobin (Hb) fractions showed an abnormal fast moving Hb component. We aimed to determine if this variant was the cause of polycythemia in this patient. A complete blood count (CBC) was performed by an automated cell counter and Hb fractions were determined by high performance liquid chromatography (HPLC). Standard stability tests and oxygen affinity evaluation were also performed. Genomic DNA was extracted from peripheral blood leukocytes using the phenol chloroform method and the entire β-globin gene was analyzed by direct sequencing. At the hematological level, no anemia or hemolysis was observed but an abnormal Hb fraction was detected using cation exchange HPLC. Molecular analysis of the β-globin gene showed heterozygosity for an AAG > ACG substitution at codon 144, resulting in a Lys→Thr amino acid replacement. We demonstrated that this is a new Hb variant with increased oxygen affinity. Its altered physiology is caused by the reduction of 2,3-diphosphoglycerate (2,3-DPG) effects, due to an amino acid substitution in the central pocket near the C-terminal of the β chain. We called this new variant Hb San Cataldo for the native city of proband.

Hemoglobin variants – pathomechanism, symptoms and diagnosis

Abstract The diagnosis of hemoglobin variants that are not any of the better-known forms of thalassemia, sickle cell, HbC, HbD, or HbE anomalies is often challenging and requires detailed knowledge of the difference in symptoms and analysis. Experience in laboratory medicine plays an important role as the range of variants is extensive and lack of expertise can result in a wrong diagnosis. Hemoglobin variants with low oxygen affinity may present cyanosis and low oxygen saturation levels, whereas variants with increased oxygen affinity show polyglobulia and concomitant complications. Differential diagnosis of methemoglobin variants requires careful assessment, which can be problematic especially in pediatric medicine. Other variants, due to their instability, can cause more or less distinct hemolysis or thalassemia syndromes depicting serious disease patterns. Clear distinction is not always possible as several symptoms are often present. Many variants are autosomal dominant inherited.

Methemoglobinemia After Isolated Lidocain Spray: A Rare Phenomenon

AbstractMethemoglobinemia is a state recognized by the increased production of met-hemoglobin, which is a form of oxidized hemoglobin, which is unable to bind oxygen. As a result the patient has a functional anemia, in which the remaining oxyhemoglobin has increased oxygen affinity, shifting the oxygen curve to left and perpetuating the impairment of oxygen delivery to tissues. Methemoglobinemia is a rare, but serious cause of hypoxemia, which can be difficult to recognize. It has been traditionally associated with the use of benzocaine class of anesthetic agents and FDA has issued warning for the use of benzocaine class of anesthetic agents for the same reason. It has been very seldom reported with the use of lidocaine class of agents alone. Through this case report we want to emphasize development of methemoglobinemia through the isolated use of lidocaine.

Mass spectrometric analysis of unknown haemoglobin fractions identified by high-performance liquid chromatography in an antenatal screening programme

AimsThe majority of haematology laboratories use automated high-performance liquid chromatography (HPLC) as a primary method in the screening of samples for haemoglobin variants and thalassaemia. HPLC detects adducted fractions, such...

Congenital familial erythrocytosis: A case report with literature review

Familial erythrocytosis is a heterogeneous group of hereditary conditions with an increased total red blood cell volume. The disease occurs in a familial pattern and follows a relatively benign course. The absence of leukocytosis and thrombocytosis differentiates it from polycythemia vera. The best characterized primary erythrocytosis is the autosomal dominant primary familial and congenital polycythemia. In most families, erythrocytosis is due to abnormal hemoglobin with increased oxygen affinity. In other families, erythrocytosis is caused by an autonomous production of erythropoietin (EPO). EPO receptor gene mutations are also responsible for erythrocytosis when no secondary causes are apparent. Presented herein is a family with familial erythrocytosis possibly due to high autonomous production of EPO, which as per the literature is the first of its kind in the Indian context.

Novel Structurally-Modified Allosteric Effectors of Hemoglobin Exhibit Superior Antisickling Properties

Sickle cell disease (SCD) continues to cause significant morbidity, mortality and healthcare disparities. Despite considerable progress in understanding the underlying pathophysiology and investigating various therapeutic strategies, novel pharmacologic approaches to ameliorate SCD continue to hold immense potential and promise, especially for patients in developing countries.Our group and others have recently renewed and refocused attention to candidate drugs that directly bind to hemoglobin (Hb) and increase oxygen (O2) affinity, preventing the fundamental pathophysiology of the disease, i.e., sickle Hb (Hb S) polymerization and red blood cell (RBC) sickling. While several candidate drugs have shown biological activity in-vitro, ex-vivo and in animal studies, their ultimate success in clinical studies was hampered by toxicity concerns and/or low oral bioavailability. Recent promising reports from a phase I/II study on 5-HMF renews optimism for this therapeutic approach.We reasoned that modifications of vanillin–a previously reported antisickling agent and food constituent without known toxicities–to enhance its efficacy, would represent a feasible approach in rationally developing clinically useful candidate drugs. Consequently, we designed and synthesized two classes of compounds: INN and TD series. The former are pyridyl derivatives of vanillin, rationalized to stereospecifically inhibit deoxy-Hb S polymer formation while increasing the fraction of the soluble oxy-Hb S in regions of low O2 tension. The TD compounds represent further modification of corresponding INN compounds (with a methoxyl group on the pyridine ring), rationalized to exhibit similar dual antisickling effects, but with enhanced direct polymer destabilization properties.We subjected a prototypical compound from each class (INN-270 and TD-7) to our battery of exploratory in-vitro assays, specifically: 1) rates of Hb S binding/modification, 2) corresponding change in O2 affinity, 3) direct inhibition of Hb S polymerization, and 4) inhibition of RBC sickling under hypoxia. We incubated 0.5, 1, or 2 mM of either INN-270 or TD-7 with RBCs from patients with homozygous SCD, under hypoxia (4% O2/96% N2 gas mixture) in a shaker-incubator at 37 ˚C for 3 h. Assays were conducted in at least three replicates utilizing different samples on different days. At the conclusion of each assay, aliquot samples (~ 10 μl each) were drawn into a fixing solution under hypoxia to preserve RBC morphology for analyses. Residual RBC suspensions were washed, hemolyzed, and subjected to: cation-exchange HPLC (to determine Hb modification); P50 analyses to establish change in O2 affinity; and temperature-dependent delay time studies to establish a delay in Hb S polymerization.Our results show that both compounds permeated RBC membranes without causing hemolysis, bound to and modified intracellular Hb at high levels in a dose dependent manner, increased O2 affinity significantly, and inhibited sickling of RBCs under hypoxia. TD-7 modified Hb S in a dose-dependent manner (to 92.3 ± 5.2 %, n=4 at 2 mM), shifted O2 equilibrium to the left (Δp50 = 45.6 ± 8.2 %, n=3 at 2 mM), and inhibited RBC sickling (by 95 -100 %, n=4). Preliminary delay time analyses also showed that at 2 mM, TD-7 increased the Hb S polymerization times from 18.1 ± 1.0 min to 24.5 ± 0.5 min. INN-270 showed a similar profile, however with a lower efficacy (at 2 mM) for Hb S modification (to ~ 75 %), Δp50 of 40.3 %, sickling inhibition by ~ 70 %, and increased delay times from 15.6 ± 0.5 min to 19.7 ± 1.0 min.We have elucidated the dual antisickling mechanism of action of INN-270 and TD-7 by X-ray crystallography. Two molecules of each compound bind to Hb via Schiff-base, and a series of hydrogen-bond/hydrophobic interactions that favor a high-O2-affinity Hb state. Importantly, the methoxyl group on the pyridine ring of TD-7 forms hydrogen-bond interactions with the surface-located αF-helix, resulting in a conformational change, possibly explaining the improved potency.Based on our results, both TD7 and INN 270 exhibited greater than a 40- and 3-fold superiority in efficacy compared to vanillin and 5-HMF, respectively. We conclude that our findings justify a prospective, structure-based approach to designing novel antisickling agents with enhanced potency. In-vitro/ex-vivo murine and human PK/PD studies are currently ongoing to help guide planned in-vivo PK/PD studies in mice. DisclosuresVenitz:Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: Consultancy. Safo:Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.

Effects of 5-hydroxymethyl-2-furfural on the volume and membrane permeability of red blood cells from patients with sickle cell disease.

The heterocyclic aldehyde 5-hydroxymethyl-2-furfural (5HMF) interacts allosterically with the abnormal form of haemoglobin (Hb), HbS, in red blood cells (RBCs) from patients with sickle cell disease (SCD), thereby increasing oxygen affinity and decreasing HbS polymerization and RBC sickling during hypoxia. We hypothesized that should 5HMF also inhibit the main cation pathways implicated in the dehydration of RBCs from SCD patients - the deoxygenation-induced cation pathway (Psickle), the Ca(2+)-activated K(+) channel (the Gardos channel) and the K(+)-Cl(-) cotransporter (KCC) - it would have a synergistic effect in protection against sickling, directly through interacting with HbS, and indirectly through maintaining hydration and reducing [HbS]. This study was therefore designed to investigate the effects of 5HMF on RBC volume and K(+) permeability in vitro. 5HMF markedly reduced the deoxygenation-induced dehydration of RBCs whether in response to maintained deoxygenation or to cyclical deoxygenation/re-oxygenation. 5HMF was found to inhibit Psickle, an effect which correlated with its effects on sickling. Deoxygenation-induced activation of the Gardos channel and exposure of phosphatidylserine were also inhibited, probably indirectly via reduced entry of Ca(2+) through the Psickle pathway. Effects of 5HMF on KCC were more modest with a slight inhibition in N-ethylmaleimide (NEM, 1 mm)-treated RBCs and stimulation in RBCs untreated with NEM. These findings support the hypothesis that 5HMF may also be beneficial through effects on RBC ion and water homeostasis.

Identification of a small molecule that increases hemoglobin oxygen affinity and reduces SS erythrocyte sickling.

Small molecules that increase the oxygen affinity of human hemoglobin may reduce sickling of red blood cells in patients with sickle cell disease. We screened 38 700 compounds using small molecule microarrays and identified 427 molecules that bind to hemoglobin. We developed a high-throughput assay for evaluating the ability of the 427 small molecules to modulate the oxygen affinity of hemoglobin. We identified a novel allosteric effector of hemoglobin, di(5-(2,3-dihydro-1,4-benzodioxin-2-yl)-4H-1,2,4-triazol-3-yl)disulfide (TD-1). TD-1 induced a greater increase in oxygen affinity of human hemoglobin in solution and in red blood cells than did 5-hydroxymethyl-2-furfural (5-HMF), N-ethylmaleimide (NEM), or diformamidine disulfide. The three-dimensional structure of hemoglobin complexed with TD-1 revealed that monomeric units of TD-1 bound covalently to β-Cys93 and β-Cys112, as well as noncovalently to the central water cavity of the hemoglobin tetramer. The binding of TD-1 to hemoglobin stabilized the relaxed state (R3-state) of hemoglobin. TD-1 increased the oxygen affinity of sickle hemoglobin and inhibited in vitro hypoxia-induced sickling of red blood cells in patients with sickle cell disease without causing hemolysis. Our study indicates that TD-1 represents a novel lead molecule for the treatment of patients with sickle cell disease.

Effects of a hemoglobin-based oxygen carrier (HBOC-201) and derivatives with altered oxygen affinity and viscosity on systemic and microcirculatory variables in a top-load rat model

The effects of a polymerized bovine hemoglobin-based oxygen carrier (HBOC) and two derivatives on arteriolar vasoactivity and tissue oxygen tension were explored by administering HBOC in a dose–response fashion to normovolemic rats. The effect of oxygen affinity (P50) and viscosity was also explored, where the P50 and viscosity of the parent compound (HBOC-201) and its modifications (MP50 and LP50A) were as follows: 40mmHg and 3.0cP (HBOC-20l); 18mmHg and 4.4cP (MP50); and 17mmHg and 12.1cP (LP50A). Anesthetized male Sprague–Dawley rats (N=32) were randomized to receive one of the HBOC solutions, and were administered four infusions that increased in concentration for each dose (2, 22, 230 and 780mg/kg, IV). Data were compared to rats receiving an equivalent volume for each of the four infusions (0.4, 0.4, 3.8, 13.1ml/kg, IV) of iso-oncotic 5.9% human serum albumin (HSA). Increasing doses of either HBOC solutions or HSA were associated with increasing MAP. Doses 3 and 4 of HBOC-201, MP50 and HSA produced significant increases in MAP, whereas similar increases began at a lower dose (Dose 2) with LP50A. There were no significant changes in arteriolar diameters at any dose for any group. Interstitial partial pressure of oxygen (ISF PO2) remained unchanged for HBOC-201, MP50 and HSA, but LP50A caused a significant decrease in ISF PO2 compared to baseline after Doses 3 and 4. In conclusion, there was no evidence that HBOC-201 would perform better with increased oxygen affinity (40 to 18mmHg) or viscosity (3.0 to 4.4cP).

The crystal structure of oxy hemoglobin from high oxygen affinity bird emu (Dromaius novaehollandiae).

Hemoglobin is an honorary enzyme, a two-way respiratory carrier, transporting oxygen from the lungs to the tissues and facilitating the return transport of carbon dioxide. Hemoglobin has high affinity for oxygen and low affinity for carbon dioxide and other substances in the arterial circulation, whereas in the venous circulation these relative affinities are upturned. The oxygen affinity of hemoglobin increases with the fall in temperature and decreases with the increase in pH and 2, 3-bisphosphoglycerate; point mutations also affect the tetrameric arrangement and alter the oxygen affinity. Though several studies have revealed the specific reasons for the adaptation of increased oxygen affinity of avian hemoglobins at high-altitudes, further structural insights on hemoglobins from high oxygen affinity species are required to understand the detailed oxygen adaptation at the molecular level. Herein, we describe the structural investigation of hemoglobin from emu (Dromaius novaehollandiae), a high oxygen affinity bird. Hemoglobin from emu was purified using anion-exchange chromatography, crystallized and determined the structure in the oxy form at a resolution of 2.3 Å; the R-factor of the model was 19.2%. The structure was compared with other oxy hemoglobins of high oxygen affinity avian species; significant changes are noted at intra-subunit contacts which provide the clues for increased oxygen affinity of emu hemoglobin.

THE STATE OF BLOOD OXYGEN-TRANSPORT FUNCTION IN PATIENTS WITH ARTERIAL HYPERTENSION AND BRONCHIAL ASTHMA

The aim of the study was to determine the condition of the blood oxygen-transport function in patients with arterial hypertension combined with bronchial asthma. 91 patients and 30 healthy individuals were examined. The blood oxygen-transport function was determined on the microaerotonometer «Syntesis-15» (Laboratory USA). The patients with arterial hypertension combined with asthma revealed an increase in oxygen affinity to hemoglobin, oxyhemoglobin dissociation curve shift to the left, an increasing degree of oxygenation of hemoglobin and the oxygen content in the venous blood. We identified the correlations between the parameters of the day monitoring of ambulatory blood pressure and blood oxygen.

Thiocarbmoylation Chemistry Based Pegylation Of αα Fumaryl-Hb Is Unique As It Does Not Induce An Increase In Oxygen Affinity: High Oxygen Affinity Pegylated Crosslinked Hbs Are Better As Oxygen Therapeutics

BackgroundPEGylated Hbs have emerged as a novel class of nohypertensive, oxygen carrying low viscosity nitric oxide producing resuscitation fluids. The limitations of PEGylation of Hb are that it induces a weakening of its interdimeric interactions and increases oxygen affinity. Besides, the influence of oxygen affinity of PEG-Hb on the tissue oxygen is still a unknown zone in the blood substitute field. In this study, we prepared two PEGylated intramolecularly crosslinked Hb and compared their performance in the extreme hemodilution model. MethodsHb-PEG adducts were prepared by incubation of PEG reagents with Hb to conjugate PEG chains to the amino groups on the surface of Hb molecule. The unreacted PEG was removed by tangential flow filtration against PBS using Minim (PAL Co.) at 4 °C. The oxygen equilibrium curves of Hb-PEG solutions were obtained using a Hemox analyzer (TCS Scientific, New Hope, PA). Extreme hemodilution: By progressive hemodilution, the hamster's systemic Hct is reduced to 18% with a non- O2 carrying colloid plasma expander (dextran 70). It is at the critical transition of Hb where O2 carrying capacity becomes limited that the study materials are introduced. This final hemodilution step is performed with the study material until an 11% hematocrit is reached, a control being generated using dextran 70. O2 delivery to the microcirculation and O2 extraction to the tissue were determined by measured by the Phosphorescence Decay (PD) Technique. ResultsThe extension arm facilitate (EAF) PEGylation as well direct PEGylation using either reductive alkylation chemistry or acylation chemistry based PEGylation of αα fumaryl-Hb generates PEG Hbs with an higher oxygen affinity (∼20 mm Hg), as compared to the oxygen affinity of 30 mm Hg for αα fumaryl-Hb. The thiocarbmoylation chemistry based PEGylation of αα-fumaryl Hb using isothiocyanate phenyl PEG 5K (ITP-PEG5K) has been investigated here. TetraPEGylated αα fumaryl-Hb [(TCP-PEG5K)4 αα-Hb] generated by thiocarbmoylation chemistry generates thiourethane linkage and exhibits an oxygen affinity (34 mm Hg), which is slightly lower than that of parent molecule (30 mm Hg). The tetraPEGylation by ITP PEG exhibits very high site selectivity to the four α-amino groups, which is the same as the reductive PEGylation. The reductive PEGylation results in a significant increase in the oxygen affinity of αα-fumaryl Hb (15 mm Hg). The oxygen affinity of (TCP-PEG 5K)4 ββ-Hb is around 8 mm Hg, which is comparable to that of ββ-Hb. Thus PEG-chains on the amino termini of crosslinked Hb with thiourethane linkage is distinct as it does not increase the oxygen affinity of crosslinjed Hb. Despite the low P50 of (TCP-PEG5K)4 αα-Hb, it neutralized the in vivo hypertensive activity of αα-fumaryl Hb. Besides, the tissue oxygenation by (TCP-PEG5K)4 αα-Hb is 3.5 mmHg, and is lower than that for affinity reductive PEGylated Hb, 5.4 mmHg. Conclusions(i) Conjugation chemistry dictates the influence on the oxygen affinity of PEGylated crosslinked Hbs and not the PEG shell, (ii) The number and amount of PEG-chains in the PEG-shell dictates the plasma expander like properties of PEG Hb; (iii) PEGylation mediated attenuation of the hypertensive activity of Hb is not a correlate of its oxygen affinity; and (iv) The tissue oxygenation PEGylated αα Hb is not correlated with oxygen affinity. Accordingly, the choice of the oxygen affinity for PEG-Hb and chemistry of PEGylation for its application as an oxygen therapeutic will be dictated by the overall efficacy of tissue oxygenation expected for a PEG Hb solution in a given clinical setting. We speculate that (TCP-PEG5KK)4 ββ-Hb will achieve a tissue oxygenation better or comparable to PEG5K2 Hb, the molecule that has given the best tissue oxygenation as an oxygen therapeutic in extreme hemodilution model so far. Disclosures:No relevant conflicts of interest to declare.

GTx011, a Potent Allosteric Modifier Of Hemoglobin Oxygen Affinity, Delays Polymerization and Prevents Sickling

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β-globin gene leading to formation of hemoglobin S (HbS). A primary and obligatory event in the molecular pathogenesis of SCD is the polymerization of deoxygenated HbS leading to sickling of red blood cells (RBCs). Prolonging the oxygenation of HbS should in principle delay polymerization relative to transit time through microcirculation, thus avoiding sickling. Discovered by Global Blood Therapeutics, GTx011 is a novel orally available small molecule that demonstrates desirable pharmacokinetics in multiple species. In addition, GTx011 binding to HbS produces concentration-dependent left shifts in partial oxygen pressure (p50) of oxygen equilibrium curves, indicating an increase in oxygen affinity. We report here that GTx011 delays in vitro polymerization and prevents sickling.GTx011 was evaluated in an adapted version of the polymerization assay described by Adachi et al. [Blood Cells (1982) 8:213-224]. Purified HbS (pre-incubated with GTx011) in 1.8 M potassium phosphate was passively de-oxygenated, followed by an induction of polymerization via temperature jump from 4°C to 37°C. Polymerization was quantified by measuring turbidity of the HbS solution under continued hypoxia. For sickling experiments, RBCs pre-incubated with GTx011 were subjected to hypoxia (pO2 of ∼30 mmHg) for 0.5 hr and subsequently imaged using a light microscope. In de-sickling experiments, RBCs were first sickled under hypoxia and then treated with GTx011 under continued hypoxia (2 hr) before acquiring images of cells. The percentage of sickled cells in each image was quantified using CellVigene software.GTx011 delayed HbS polymerization in a dose-dependent manner. The delay profile was similar to that of CO-liganded HbA, a well characterized intracellular inhibitor of HbS polymerization. Moreover, GTx011 prevented sickling of RBCs under hypoxia suggesting that GTx011 has the ability to prevent intracellular HbS polymerization. It should be noted that in this experimental system, RBCs were exposed to hypoxia for a relatively longer period of time than typical RBC transit times through microcirculation. Interestingly, GTx011 also reversed sickling of pre-sickled RBCs. The ability of GTx011 to de-sickle RBCs suggests that it may promote transition from the low affinity HbS (T-state HbS) into its high affinity conformer (R-state HbS), a form that is not incorporated into HbS polymers. Thus, by stabilizing the oxy (R) conformation of HbS, GTx011 has the potential to be a therapeutic agent for prevention of HbS polymerization and sickling in SCD patients.Table 1AssayPolymerizationSicklingDe-sicklingUnitDTcpd-DTHbS(min)(% sickled)(% sickled)[HbS]50 µM∼1 mM (20% HCT)∼1 mM (20% HCT)[Cpd]25 µM50 µM100 µM1 mM2 mM5 mM1 mM2 mM5 mMGTx0113.912.822.94133285734315-HMF11.86.3766550979689No Cpd000858585969696Table 2AssayPolymerizationUnitDTHbS/HbA-DTHbS(min)[Hb]50 µM% HbA-CO20%30%40%HbA-CO2.311.816.8 Disclosures:Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.

The Anti-Sickling Agent Aes-103 Decreases Sickle Erythrocyte Fragility, Hypoxia-Induced Sickling and Hemolysis In Vitro

BackgroundThe polymerization of hemoglobin S results in red cell morphological changes and fragility, which promotes hemolysis. Aes-103 (5-hydroxymethylfurfural, 5-HMF) is a clinical-stage candidate anti-sickling agent that binds to alpha subunits of hemoglobin, increases its oxygen affinity and stabilizes the R-state. In vitro at, millimolar levels, it inhibits hypoxia-induced sickling and in vivo protects sickle cell mice against hypoxia-induced death. We have found in vitro that Aes-103 increases oxygen affinity of red cells from healthy control subjects and from patients with sickle cell disease either on or off hydroxyurea. We further investigated the ability of Aes-103 to protect red cells from patients with sickle cell anemia from a variety of effects of sickling.First, we used transmission electron microscopy (TEM) to investigate fiber formation in human sickle blood incubated with 5 mM Aes-103 for one hour at 37° prior to deoxygenation (2% oxygen for two hours) and fixed with glutaraldehyde, and embedded by standard techniques. Semi-quantitative analysis of the images showed that the addition of Aes-103 was able to significantly reduce the percentage of cells displaying elongated fibers (p=0.048). Furthermore, we confirmed that Aes-103 incubation prior to hypoxia also reduces morphological sickling as quantified by sickle imaging flow cytometry (P=0.0027). Fiber formation was associated with morphological sickling (R=0.522, P=0.027).Second, we investigated the effects of Aes-103 on sickle erythrocyte fragility. In vitro shear stress induced by rotation on a vertical rotator at 21 revolutions per minute for 3 hours promoted hemolysis in blood samples from patients with SCA (free hemoglobin 29.4 ± 3.4 vs. 8.4 ± 0.9 mM, p<0.001, n=10). Addition of Aes-103 at increasing concentrations for one hour prior to testing reduced the extent of shear-stress induced hemolysis, ranging from decreases of 15% at 1 mM to 28% at 2mM to 37% at 5 mM (p<0.001). Interestingly, although shear stress promoted less hemolysis in blood samples from healthy controls, Aes-103 reduced hemolysis in healthy control blood to a comparable extent, suggesting a red cell stabilizing mechanism distinct from any anti-sickling effect.Third, we evaluated the degree of hemolysis associated with erythrocyte sickling induced by deoxygenation. Hypoxia (2% oxygen for 2 hours) induced hemolysis, and the severity of hemolysis was reduced by the addition of Aes-103 for one hour (prior to hypoxia) at increasing concentrations up to 5 mM (free hemoglobin 31.3 ± 2.9 vs. 21.3 ± 2.8 mM, p = .002, n=11).In summary, treatment of sickle blood with Aes-103 in vitro, decreases fiber formation, increases oxygen affinity of sickle red cells, reduces sickle cell mechanical fragility, and reduces hemolysis caused by hypoxia-induced sickling. Aes-103 merits investigation as a potential alternative or adjunct to hydroxyurea as a treatment for sickle cell disease. We have completed a phase I clinical trial of oral Aes-103 at the NIH Clinical Center, and a separate abstract reports its apparent safety and tolerability. A phase 2 twenty-eight day study is being initiated in the U.K. in adults with sickle cell anemia. Disclosures:No relevant conflicts of interest to declare.

Hemoglobin Analyses in The Netherlands Reveal More Than 80 Different Variants Including Six Novel Ones

More than 20 000 blood samples of individuals living in The Netherlands and suspected of hemolytic anemia or diabetes were analyzed by high resolution cation exchange high performance liquid chromatography (HPLC). Besides common disease-related hemoglobins (Hbs), rare variants were also detected. The variant Hbs were retrospectively analyzed by capillary zone electrophoresis (CZE) and by isoelectric focusing (IEF). For unambiguous identification, the globin genes were sequenced. Most of the 80 Hb variants detected by initial screening on HPLC were also separated by capillary electrophoresis (CE), but a few variants were only detectable with one of these methods. Some variants were unstable, had thalassemic properties or increased oxygen affinity, and some interfered with Hb A2 measurement, detection of sickle cell Hb or Hb A1c quantification. Two of the six novel variants, Hb Enschede (HBA2: c.308G > A, p.Ser103Asn) and Hb Weesp (HBA1: c.301C > T, p.Leu101Phe), had no clinical consequences. In contrast, two others appeared clinically significant: Hb Ede (HBB: c.53A > T, p.Lys18Met) caused thalassemia and Hb Waterland (HBB: c.428C > T, pAla143Val) was related to mild polycytemia. Hb A2-Venlo (HBD: c.193G > A, p.Gly65Ser) and Hb A2-Rotterdam (HBD: c.38A > C, p.Asn13Thr) interfered with Hb A2 quantification. This survey shows that HPLC analysis followed by globin gene sequencing of rare variants is an effective method to reveal Hb variants.

A New High Affinity Variant Hb Aurillac (β141Leu→Val)

Hemoglobin (Hb) variant β141(H19)Leu→Val (HBB:c.424C>G), one of the two mutations defining Hb Kochi [the other one being β144(HC1)Lys-Tyr-His→0 (HBB:c.433A>T)], was found as an isolated mutation. In contrast to what was suggested for Hb Kochi, the new variant was not clinically silent. It displayed increased oxygen affinity and was associated with mild erythrocytosis.

HBOC Vasoactivity: Interplay Between Nitric Oxide Scavenging and Capacity to Generate Bioactive Nitric Oxide Species

Despite many advances in blood substitute research, the development of materials that are effective in maintaining blood volume and oxygen delivery remains a priority for emergency care and trauma. Clinical trials on hemoglobin (Hb)-based oxygen carriers (HBOCs) have not provided information on the mechanism of toxicity, although all commercial formulations have safety concerns. Specifically, it is important to reconcile the different hypotheses of Hb toxicity, such as nitric oxide (NO) depletion and oxidative reactions, to provide a coherent molecular basis for designing a safe HBOC. HBOCs with different sizes often exhibit differences in the degree of HBOC-induced vasoactivity. This has been attributed to differences in the degree of NO scavenging and in the extent of Hb extravasation. Additionally, it is appears that Hb can undergo reactions that compensate for NO scavenging by generating bioactive forms of NO. Engineering modifications to enhance bioactive NO production can result in diminished oxygen delivery by virtue of increased oxygen affinity. This strategy can prevent the HBOC from fulfilling the intended goal on preserving oxygenation; however, the NO production effects will increase perfusion and oxygen transport. Hb modifications influence NO scavenging and the capacity of certain HBOCs to compensate for NO scavenging through nitrite-mediated reactions that generate bioactive NO. Based on the current understanding of these NO-related factors, possible synthetic strategies are presented that address how HBOC formulations can be prepared that: (i) effectively deliver oxygen, (ii) maintain tissue perfusion, and (iii) limit/reverse underlying inflammation within the vasculature.

A non‐electrolyte haemolysis assay for diagnosis and prognosis of sickle cell disease

Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.

Analysis of oxygen affinity in aquatic amphibian; homology modelling of the major Haemoglobin component HbA1 from the African clawed frog (Xenopus laevis, Anura)

The homology model of major haemoglobin component HbA1 of the African Clawed Frog was predicted using the pigeon (Columba livia) haemoglobin as a template. The model was built with the help of MODELLER9v8. The models were evaluated with ProSA and PROCHECK. In X. laevis Gln38α is unable to form a hydrogen bond with β97His or β99Asp, which is responsible for the increase in oxygen affinity of the Xenopus HbA1. The hydrogen bond between α34Thr and β124Pro, which stabilises the deoxy state of the haemoglobin, was absent in X. laevis. Hence it is predicted that the HbA1 component of X. laevis has higher oxygen affinity.

Glutathionylation Induced Structural Changes in Oxy Human Hemoglobin Analyzed by Backbone Amide Hydrogen/Deuterium Exchange and MALDI-Mass Spectrometry

Glutathionyl hemoglobin, a post-translationally modified form of hemoglobin, has been reported to serve as a marker of oxidative stress in several clinical conditions. This modification causes perturbations in the hemoglobin functionality by increasing oxygen affinity and reducing cooperativity. Moreover, glutathionylation of sickle hemoglobin was reported to lead to a significant reduction in the propensity of sickling of erythrocytes. The root cause of the above functional abnormality is not known in detail, as the crystal structure of the molecule is yet to be discovered. In this study, we investigated the effects of glutathionylation on quaternary structure of hemoglobin using hydrogen/deuterium exchange (H/DX) based mass spectrometry. H/DX kinetics of nine peptides from α and β globin chains of hemoglobin were analyzed to understand the conformational change in deoxy to oxy transition of normal hemoglobin and structural perturbations associated with glutathionylation of oxy hemoglobin. Significant structural changes brought about by the glutathionylation of oxy hemoglobin were observed in the following regions of globin chains: β86-102, β1-14, α34-46, β32-41, β130-146, β115-129, β73-81. Isotope exchange kinetics monitored through mass spectrometry is a useful technique to understand structural perturbation on post-translational modification of proteins in solution phase.