Low Oxygen Affinity Research Articles

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.

Phthalides serve as potent modulators to boost fetal hemoglobin induction therapy for β-hemoglobinopathies

AbstractFetal hemoglobin (HbF) induction therapy has become the most promising strategy for treating β-hemoglobinopathies, including sickle-cell diseases and β-thalassemia. However, subtle but critical structural difference exists between HbF and normal adult hemoglobin (HbA), which inevitably leads to reduced binding of the endogenous modulator 2,3-bisphosphoglycerate (2,3-BPG) to HbF and thus increased oxygen affinity and decreased oxygen transport efficiency of HbF. We combined the oxygen equilibrium experiments, resonance Raman (RR) spectroscopy, and molecular docking modeling, and we discuss 2 phthalides, z-butylidenephthalide and z-ligustilide, that can effectively lower the oxygen affinity of HbF. They adjust it to a level closer to that of HbA and make it a more satisfactory oxygen carrier for adults. From the oxygen equilibrium curve measurements, we show that the 2 phthalides are more effective than 2,3-BPG for modulating HbF. The RR spectra show that phthalides allosterically stabilize the oxygenated HbF in the low oxygen affinity conformation, and the molecular docking modeling reveals that the 2 chosen phthalides interact with HbF via the cleft around the γ1/γ2 interface with a binding strength ∼1.6 times stronger than that of 2,3-BPG. We discuss the implications of z-butylidenephthalide and z-ligustilide in boosting the efficacy of HbF induction therapy to mitigate the clinical severities of β-hemoglobinopathies.

Characterization of deletional and non-deletional alpha globin variants in a large cohort from Spain between 2009 and 2014.

The hemoglobinopathies are a group of disorders passed down through families (inherited) in which there is abnormal production or structure of the hemoglobin molecule. They are among the most common inherited diseases around the world. Those that produce abnormal hemoglobin are called structural hemoglobinopathies while thalassemia is another type of disorder that is caused by a defect in the gene production of the globin chains. In a study ambispective comprising 1623 patients, 153 subjects showed an abnormal hemoglobin and 1470 with hypochromic and microcytic anemia, and of these 1470, 23 patients were studied for simultaneously α-thalassemias and structural hemoglobinopathies. Among the α-thalassaemia cases, 1282 cases (87.2%) were deletional α-thalassemia, 172 cases (11.7%) were non-deletional α-thalassemia, and 16 cases (1.1%) were deletional and non-deletional α-thalassamias simultaneously. Thus, approximately 12% of the cases were non-deletional α-thalassaemia. Clinical diagnosis, only 19 severe cases (1 hydrops fetalis and 18 instances of Hb H disease), 1200 thalassamias traits, and 160 thalassaemia silent carriers were recorded within the α-thalassaemia. Regarding structural hemoglobinopathies, there were only 2 cases of hemoglobinopathies with low oxygen affinity and 1 case of hemoglobin M; the remaining 150 were silent hemoglobinopathies. Non-deletional α-thalassaemia represented 12% of all α-thalassemias in our region; the most common deletion in our area was the 3.7-kb deletions, followed by Asian --(SEA) and --(FIL). The alterations responsible for non-deletional α-thalassaemia are most represented by the Hph and Hb Groene Hart and, in the case of structural hemoglobinopathies, Hb Le Lamentin and Hb J-Paris.

Micro-Second X-Ray Single Molecule Dynamics of Allosteric Twisting Motions in Hemoglobin

Allosteric structural changes underlie the biological functions of many proteins. The best studied is the tense (T) to relaxed (R) state transition in hemoglobin (Hb), which allows for efficient oxygen transport. These two end structures have been solved by crystallography, but little is known about the transition pathway and motions between them. Here we use Diffracted X-ray Tracking (DXT) to monitor single molecule allosteric dynamics in Hb. DXT allows us to monitor the protein motions at high time resolution by tracking diffracted spots from a gold-nanocrystal attached to the protein. X-ray from SPring-8 BL40XU with pink beam was used for the DXT measurements. Data from two rotational directions of the protein molecule, those concerning tilting angle θ and twisting angle χ, are detected. Hb trout III extracted from rainbow trout was used as the sample, which exhibits a remarkable Root effect. It is expected that at pH 8 Hb trout III would exist as an ensemble of the R states with a high oxygen affinity, while at pH 6 it would exist as the T state with a low affinity for oxygen, which is one-thousandth of that of the R state. These unique properties allow us to select conformational populations of Hb by simply adjusting the pH values of the solvents used. Basic time resolution of the system was 100μs/frame. However, we have confirmed that we can observe much faster motions in a solvent containing 30% glycerol at low temperature, where molecular motions of Hb are markedly decelerated. As a result of the DXT observations, we succeeded to clearly detect motions in different twisting direction at a specific moving speed (5-10mard/ms).

Hepcidin Predicts Decrease in Erythroid Expansion in Sickle Cell Disease Patients Treated with Inhaled Steroids

Sickle cell disease (SCD) is a recessively inherited hemoglobin disorder; the most common and severe form is a consequence of homozygous βS mutation. High concentration of hemoglobin S damages RBC membranes, leading to hemolysis, vaso-occlusion, and inflammation, which together result in anemia, recurrent painful crises, and multiple end-organ damage (i.e. brain, kidney, lung, and bone). Anemia in SCD is multi-factorial. Causes include hemolysis, ineffective erythropoiesis, impaired iron utilization, insufficient erythropoietin responsiveness, and low oxygen affinity [Sherwood Blood 1986]. RBC transfusions are used to ameliorate symptoms and complications but indications are not clearly defined for adults. We postulate that impaired iron utilization in SCD is a consequence of complex regulation of hepcidin, the main hormone regulating circulating and systemic iron. We aim to explore correlation between hepcidin, inflammation and erythropoiesis to enhance our understanding of iron metabolism, hepcidin regulation, and pathophysiology of anemia in SCD. Ultimately this may yield more specific indications for RBC transfusion, and pave the way for novel therapeutic approaches.High hepcidin results in the sequestration of iron within macrophages and prevents further iron absorption in the gastrointestinal tract. Hepcidin expression is enhanced by iron and inflammation and suppressed by hypoxia and erythropoiesis. Distinguishing the relative contribution of these competing effects on hepcidin in SCD is complicated. For example, adult SCD patients in steady state have significantly lower hepcidin relative to heterozygous controls and do not correlate with markers of inflammation and erythropoiesis [Kroot Haematologica 2009]. In a randomized, placebo-controlled trial, inhaled steroids (mometasone) were administered to non-asthmatic SCD patients [Glassberg Am J Hematol 2017]. The results demonstrate improved pain scores and reduced hemolysis in the treated group although the mechanism of this effect is not known. In a subsequent analysis, markers of inflammation are suppressed in the treated group, correlating with macrophage activation. We thus evaluate hepcidin expression in stored serum samples from this trial of SCD patients treated with inhaled steroids or placebo. Samples before and after treatment were analyzed. Serum hepcidin quantification was performed with Hepcidin-25 ELISA kit (Intrinsic Lifesciences LLC). In addition, we evaluate if and how hepcidin levels correlate with pro-inflammatory markers measured with olink inflammation assay.Serum hepcidin concentration is not different in steroid- vs. placebo-treated SCD patients before or after treatment and no significant difference is observed in the hepcidin ratio after:before treatment between the two groups (Fig 1a). However, in the steroid group, hepcidin concentrations are increased in a higher proportion of patients although the difference does not reach statistical significance (Fig 1b). We thus hypothesize that hepcidin levels may be a marker of increased erythropoiesis relative to inflammation in SCD. Both IL-6 (Fig 2a) and IL-10 (Fig 2b), cytokines known to induce hepcidin expression during inflammation, decrease after treatment with steroids relative to placebo, and neither correlates with hepcidin concentration after treatment (Table I). Hepcidin is however inversely correlated with adenosine deaminase (ADA) and hemoglobin S and directly correlative with fibroblast growth factor 23 (FGF23), monocyte chemoattractant protein 1 (MCP1), and hemoglobin F in both placebo- and steroid-treated groups (Table I), potentially suggesting that hepcidin inversely correlates with expanded erythropoiesis. We thus use the newly available human ELISA kit for erythroferrone (ERFE) (Intrinsic Lifesciences LLC), a recently identified erythroid regulator of hepcidin [Kautz Nat Gen 2014], to evaluate correlation with hepcidin. Our results demonstrate that serum ERFE concentration decreases in steroid- (Fig 3a) but not placebo-treated SCD patients (Fig 3b), and no correlation with hepcidin is observed. Taken together, these data for the first time demonstrate a decrease in erythropoiesis and erythroid-regulation-induced hepcidin suppression in inhaled-steroid-treated SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Crystal Structure Analysis of Great Cormorant (Phalacrocorax carbo) Hemoglobin to Understand its High Oxygen Affinity Characteristics by Special Structural Features.

Hemoglobin (Hb) subunits are composed of the specific functional prosthetic group "heme'' and a protein moiety "globin". Bird Hbs are functionally similar to mammalian Hbs but they are structurally dissimilar with mammalian. The insufficient structural studies on avian Hbs limit us to understand their degree of adaptation to such critical environments. The Great Cormorant (GCT) can fly and swim, the dual characteristic of GCT leads to study the sturcture of hemoglobin. To determine the crystal structure of Great Cormorant Hemoglobin and to compare its three dimensional structure with other high and low oxygen affinity hemoglobin species to understand its characteristic features of high oxygen affinity. The GCT hemoglobin has been purified, crystallized and data sets were processed using iMosflm. The integrated data has been solved using Molecular replacement method using Graylag hemoglobin (1FAW) as the template. The structure has been deposited in Protein Data Bank with PDB code: 3WR1. In order to characterize the tertiary and quaternary structural differences, the structure of cormorant hemoglobin is compared with GLG, BHG and human Hb. The larger variation observed between GCT and human Hb indicates that GCT Hb differs remarkably from human. The α1β1 interface of Great cormorant Hb is similar to bar-headed goose Hb with few amino acid substitutions. It has been found that the interaction which is common among avian hemoglobins (α119 Pro- β55Leu) is altered by Ala 119 in GCT. This intra-dimer contact (α119 Pro - β 55 Leu) disruption leads to high oxygen affinity in BGH Hb. In cormorant, GLG and human the proline is unchanged but interestingly, in cormorant Hb, the β55 position was found to be Thr instead of Leu. Similar kind of substitutions (β 55 Leu - Ser) observed in Andean goose Hb structure leads to elevated oxygen affinity between Hb-O2. To our surprise, such type of substitution at β 55 (Thr) in cormorant Hb confirms that it is comparable with Andean goose Hb structure. Thus the sequence, structural differences at alpha, beta heme pocket and interface contacts confirms that GCT adopts high oxygen affinity conformation. The three dimensional structure of Great cormorant hemoglobin has been investigated to understand its unique structural features to adopt during hypoxia condition. By comparing the sequence and overall structural similarities with high and low oxygen affinity species, it appears that GCT has more possibilities to subsist with low oxygen demand.

Template Assisted Nucleation of Cobalt and Gold Nano-clusters on an Ultrathin Iron Oxide Film

Being the basic building blocks for nano-magnetic and nano-catalytic devices, regular arrays of nano-clusters play a crucial role in modern nanotechnology. One of the possible fabrication methods of periodic nanostructures consists in exploiting nano-patterned substrates as templates for the self-assembly of the deposited atoms. Here, we have investigated the templating properties of a Moire superlattice formed at the interface between a FeO(111)-like ultrathin film and a Ni/Fe(001) substrate. Co and Au, representative of elements with high and low oxygen affinity, respectively, have been deposited on the iron oxide film. Scanning tunneling microscopy reveals that Co nucleates preferentially along the corrugated regions of the Moire superstructure, forming stripes with high aspect ratio. On the other hand, Au atoms nucleate randomly distributed three-dimensional islands on the FeO(111) surface.

Resuscitation from Hemorrhagic Shock with High Molecular Weight Polymerized Hemoglobin

BackgroundHemoglobin (Hb) based oxygen carriers (HBOCs) have been proposed as an alternative to red blood cells (RBCs) for transfusion medicine. Lack of mechanistic analysis lead to clinical evaluation of low molecular weight (MW) HBOCs, which failed in phase III clinical trials due to a myriad of side effects not seen in pre‐clinical trials. Increasing the MW of HBOCs can decrease these side effects by confining HBOCs to the vascular space and increasing nitric oxide generation. HBOCs are advantageous to blood due to their non‐immunogenic properties and potential to use non‐human hb sources, among others. In this study we compare the efficacy of fresh blood, freshly synthesized polymerized bovine hb (PolybHb), and PolybHb stored for 2 years in restoring cardiac function after hemorrhagic shock.MethodsPolybHb was synthesized in the low oxygen affinity (T) state with a 35:1 ratio of glutaraldehyde to bovine Hb, and then subjected to 8–9 cycles of diafiltration. This resulted in a PolybHb solution containing only polymerized Hb molecules < 0.2 uM and >500kDa. PolybHb was then frozen at −80°C and stored until used. Fresh red blood cells were prepared from the blood removed during hemorrhage by centrifugation at 1000g for 7 minutes, removal of the buffy coat, hemoconcentration to a hematocrit of 50%. Rats (200–250g) were hemorrhaged for 50% of their blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre‐hemorrhage with fresh autologous blood (fresh blood), PolybHb generated within 3 months of the study (PolybHb 2017), or PolybHb generated two years prior to the study (PolybHb 2015).ResultsShock resulted in impaired oxygen delivery and cardiac function. Resuscitation with fresh blood, PolybHb 2017, and PolybHb 2015 restored cardiac function and systemic vascular resistance. Slightly more PolybHb was infused before restoration of 90% pre‐hemorrhage MAP was achieved, but the volume was not statistically significant. Likely as a result of the higher resuscitation volume, stroke volume and stroke work increased more in animals resuscitated with PolybHb than fresh autologous blood. PolybHb did not impair cardiac contractility nor cause excessive vasoconstriction.ConclusionsStudies indicate that PolybHb is as efficacious as fresh autologous blood in restoring cardiac function in rats after hemorrhagic shock. Additionally, PolybHb can be safely stored at −80°C for two years and retain efficacy, unlike red blood cells.Support or Funding InformationNIH Grants from the National Heart Lung and Blood Institute, T32‐HL105373, P01‐HL110900, R01‐HL126945This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High performance perpendicular magnetic tunnel junction with Co/Ir interfacial anisotropy for embedded and standalone STT-MRAM applications

High volume spin transfer torque magnetoresistance random access memory (STT-MRAM) for standalone and embedded applications requires a thin perpendicular magnetic tunnel junction (pMTJ) stack (∼10 nm) with a tunnel magnetoresistance (TMR) ratio over 200% after high temperature back-end-of-line (BEOL) processing up to 400 °C. A thin reference layer with low magnetic moment and strong perpendicular magnetic anisotropy (PMA) is key to reduce the total thickness of the full pMTJ stack. We demonstrated strong interfacial PMA and a perpendicular Ruderman-Kittel-Kasuya-Yosida exchange interaction in the Co/Ir system. Owing to the additional high PMA at the Ir/Co interface in combination with a conventional CoFeB/MgO interface in the Ir/Co/Mo/CoFeB/MgO reference layer, the full film pMTJ showed a TMR ratio over 210% after annealing at 400 °C for 150 min. The high TMR ratio can be attributed to the thin stack design by combining a thin reference layer with the efficient compensation by a thin pinned layer. The annealing stability may be explained by the absence of solid solution in the Co-Ir system and the low oxygen affinity of Mo in the reference layer and the free layer. High device performance with a TMR ratio over 210% was also confirmed after subjecting the patterned devices to BEOL processing temperatures of up to 400 °C. This proposed pMTJ design is suitable for both standalone and embedded STT-MRAM applications.

Engineering oxidative stability in human hemoglobin based on the Hb providence (βK82D) mutation and genetic cross-linking.

Previous work suggested that hemoglobin (Hb) tetramer formation slows autoxidation and hemin loss and that the naturally occurring mutant, Hb Providence (HbProv; βK82D), is much more resistant to degradation by H2O2 We have examined systematically the effects of genetic cross-linking of Hb tetramers with and without the HbProv mutation on autoxidation, hemin loss, and reactions with H2O2, using native HbA and various wild-type recombinant Hbs as controls. Genetically cross-linked Hb Presbyterian (βN108K) was also examined as an example of a low oxygen affinity tetramer. Our conclusions are: (a) at low concentrations, all the cross-linked tetramers show smaller rates of autoxidation and hemin loss than HbA, which can dissociate into much less stable dimers and (b) the HbProv βK82D mutation confers more resistance to degradation by H2O2, by markedly inhibiting oxidation of the β93 cysteine side chain, particularly in cross-linked tetramers and even in the presence of the destabilizing Hb Presbyterian mutation. These results show that cross-linking and the βK82D mutation do enhance the resistance of Hb to oxidative degradation, a critical element in the design of a safe and effective oxygen therapeutic.

Cardiac performance is limited by oxygen delivery to the mitochondria in the crystalloid-perfused working heart.

The left ventricular working, crystalloid-perfused heart is used extensively to evaluate basic cardiac function, pathophysiology, and pharmacology. Crystalloid-perfused hearts may be limited by oxygen delivery, as adding oxygen carriers increases myoglobin oxygenation and improves myocardial function. However, whether decreased myoglobin oxygen saturation impacts oxidative phosphorylation (OxPhos) is unresolved, since myoglobin has a much lower affinity for oxygen than cytochrome c oxidase (COX). In the present study, a laboratory-based synthesis of an affordable perfluorocarbon (PFC) emulsion was developed to increase perfusate oxygen carrying capacity without impeding optical absorbance assessments. In left ventricular working hearts, along with conventional measurements of cardiac function and metabolic rate, myoglobin oxygenation and cytochrome redox state were monitored using a novel transmural illumination approach. Hearts were perfused with Krebs-Henseleit (KH) or KH supplemented with PFC, increasing perfusate oxygen carrying capacity by 3.6-fold. In KH-perfused hearts, myoglobin was deoxygenated, consistent with cytoplasmic hypoxia, and the mitochondrial cytochromes, including COX, exhibited a high reduction state, consistent with OxPhos hypoxia. PFC perfusate increased aortic output from 76 ± 6 to 142 ± 4 ml/min and increased oxygen consumption while also increasing myoglobin oxygenation and oxidizing the mitochondrial cytochromes. These results are consistent with limited delivery of oxygen to OxPhos resulting in an adapted lower cardiac performance with KH. Consistent with this, PFCs increased myocardial oxygenation, and cardiac work was higher over a wider range of perfusate Po2. In summary, heart mitochondria are limited by oxygen delivery with KH; supplementation of KH with PFC reverses mitochondrial hypoxia and improves cardiac performance, creating a more physiological tissue oxygen delivery. NEW & NOTEWORTHY Optical absorbance spectroscopy of intrinsic chromophores reveals that the commonly used crystalloid-perfused working heart is oxygen limited for oxidative phosphorylation and associated cardiac work. Oxygen-carrying perfluorocarbons increase myocardial oxygen delivery and improve cardiac function, providing a more physiological mitochondrial redox state and emphasizing cardiac work is modulated by myocardial oxygen delivery.

Detection of Hb Rothschild HBB: c.[112T>A or 112T>C], Through High Index of Suspicion on Abnormal Pulse Oximetry

We describe a case with a low oxygen affinity hemoglobin (Hb) variant who presented with cyanosis in the absence of cardiopulmonary disease. The patient, a 27-year-old pregnant female (P1G2), complained of a productive cough and bluish discoloration of the lips that started 3 days prior to seeking attention. She had no previous episodes and has generally been in good health. A positive family history of cyanosis was obtained in one sibling. Systematic examination, notably the cardiorespiratory system, revealed no abnormalities. The arterial Hb oxygen saturation (SpO2) on pulse oximetry was 81.0% and Hb separation studies revealed an Hb variant identified as Hb Rothschild [β37(C3)Trp→Arg] (HBB: c.[112 T>A or 112 T>C]) by gene sequencing. The amino acid substitution (Trp→Arg) is an important contact point at the α1β2 interface and favors a T-quaternary state of the Hb tetramer. This leads to a low oxygen affinity state, which results in premature release of oxygen and drop in oxygen saturation. In the absence of cardiopulmonary disease, a decreased oxygen saturation reading, with or without cyanosis, should arouse suspicion for a possible dysHb.

Modulation of hemoglobin dynamics by an allosteric effector.

Hemoglobin (Hb) is an extensively studied paradigm of proteins that alter their function in response to allosteric effectors. Models of its action have been used as prototypes for structure‐function relationships in many proteins, and models for the molecular basis of its function have been deeply studied and extensively argued. Recent reports suggest that dynamics may play an important role in its function. Relatively little is known about the slow, correlated motions of hemoglobin subunits in various structural states because experimental and computational strategies for their characterization are challenging. Allosteric effectors such as inositol hexaphosphate (IHP) bind to both deoxy‐Hb and HbCO, albeit at different sites, leading to a lowered oxygen affinity. The manner in which these effectors impact oxygen binding is unclear and may involve changes in structure, dynamics or both. Here we use neutron spin echo measurements accompanied by wide‐angle X‐ray scattering to show that binding of IHP to HbCO results in an increase in the rate of coordinated motions of Hb subunits relative to one another with little if any change in large scale structure. This increase of large‐scale dynamics seems to be coupled with a decrease in the average magnitude of higher frequency modes of individual residues. These observations indicate that enhanced dynamic motions contribute to the functional changes induced by IHP and suggest that they may be responsible for the lowered oxygen affinity triggered by these effectors.

Hb Presbyterian (HBB: c.327C>G) in a Nicaraguan Family

Hemoglobin (Hb) is the protein responsible for oxygen transportation. It is a tetrameric protein comprising two α- and two β-globin subunits. In the literature, a large number of mutations in the α- and β-globin genes have been documented. Among these mutations, Hb Presbyterian (HBB: c.327 C>G), is a naturally occurring mutant exerting low oxygen affinity. The C to G exchange (AAC>AAG) at codon 108 of the β-globin gene results in the substitution of asparagine by lysine. Here, we document the identification of HBB: c.327 C>G in a 6-year-old female patient and her father from Nicaragua and Cuba, respectively. The presence of the abnormal Hb was confirmed by cellulose acetate electrophoresis, high performance liquid chromatography (HPLC) and genomic DNA sequencing. The β-globin gene sequences for both, father and daughter, disclosed the heterozygous mutation at codon 108 to be Hb Presbyterian or HBB: c.327 C>G. The mutant Hb was previously reported in four families from North America, Germany, Japan and Spain, respectively. This is the fifth family carrying HBB: c.327 C>G described to date and the first report from Latin America.

New developments in anti-sickling agents: can drugs directly prevent the polymerization of sickle haemoglobin invivo?

The hallmark of sickle cell disease is the polymerization of sickle haemoglobin due to a point mutation in the β-globin gene (HBB). Under low oxygen saturation, sickle haemoglobin assumes the tense (T-state) deoxygenated conformation that can form polymers, leading to rigid erythrocytes with impaired blood vessel transit, compounded or initiated by adhesion of erythrocytes to endothelium, neutrophils and platelets. This process results in vessel occlusion and ischaemia, with consequent acute pain, chronic organ damage, morbidity and mortality. Pharmacological agents that stabilize the higher oxygen affinity relaxed state (R-state) and/or destabilize the lower oxygen affinity T-state of haemoglobin have the potential to delay the sickling of circulating red cells by slowing polymerization kinetics. Relevant classes of agents include aromatic aldehydes, thiol derivatives, isothiocyanates and acyl salicylates derivatives. The aromatic aldehyde, 5-hydroxymethylfurfural (5-HMF) increases oxygen affinity of sickle haemoglobin and reduces hypoxia-induced sickling invitro and protects sickle cell mice from effects of hypoxia. It has completed pre-clinical testing and has entered clinical trials as treatment for sickle cell disease. A related molecule, GBT440, has shown R-state stabilization and increased oxygen affinity in preclinical testing. Allosteric modifiers ofhaemoglobin as direct anti-sickling agents target the fundamental pathophysiological mechanism of sickle cell disease.

Sintering atmospheres effects on newly developed low-alloy manganese steel powders for structural applications

Alloying elements with a low affinity for oxygen are used in powder metallurgy to satisfy a large extent of requirements, especially on mechanical properties. On the other hand these elements are often expensive, so special attention is being paid to alloying with cheaper elements which have a stronger hardening effect (e.g. Cr, Mn and Si). When combined with traditional elements like copper, molybdenum and nickel, it is possible to optimize the properties of sintered materials for structural applications. Manganese is the cheapest alloying element for many iron-based alloys, but its high affinity for oxygen makes it difficult to incorporate this element into P/M steels; as a result it is not widely used in powder metallurgy. Two alternative compositions of Cu-Ni-Mo alloy system were selected, one with low nickel ( 2 -H 2 -based atmospheres with an increasing content of methane to investigate the effects of carbon potential variation on mechanical behavior and dimensional change of sintered parts. Furthermore, sintering treatments in inert atmosphere, endogas and endogas/methane were studied. Microstructural investigations and complete mechanical characterization were carried out in order to relate properties and constituent phases to atmosphere composition.

Interactions between a Classical Allosteric Protein and a Strong Effector Revisited

According to the Two-State Model of Monod-Wyman-Changeux, the allosteric regulation in human tetrameric hemoglobin (Hb) is achieved upon binding of heterotrophic effectors, such as 2,3-bisphosphoglycerate (BPG), to the allosteric site located between the beta subunits of unliganded, low oxygen affinity T-structure Hb. As a result, the new complex exhibits an additional decrease in the affinity for oxygen when compared with Hb in the absence of BPG. On the other hand, liganded Hb in the high oxygen affinity R-structure does not bind BPG, and thus its oxygen affinity remains unaffected.We have studied the interactions between inositol hexakisphosphate (IHP), an allosteric effector stronger that DPG, and Hb, both in the liganded and unliganded forms, by isothermal titration calorimetry (ITC) and oxygen binding measurements in the equimolar range at pH 7.0 and 15°C. For the liganded “R”-structure, we chose the cyanmetHb derivative (Hb+CN-), and for the unliganded “T”-structure, the nickel-porphyrin Hb (NiHb). ITC experiments showed that IHP binds to both tetrameric derivatives in equimolar amount and with relatively high affinity.Tertiary/quaternary structural perturbations introduced systematically into Hb+CN- by removal of specific amino acid residues suggested that the IHP binding site is identical to that exhibited by NiHb, i.e., between the beta subunits. Under these conditions, we have not found any evidence that suggests IHP bind between the alpha subunits, or in the central cavity, as recent reports have suggested.At pH 8.2, we observed that while unliganded Hb with equimolar amounts of IHP remained practically invariable, the addition of above 100-fold molar excess indeed produced a pronounced decrease in the affinity for oxygen, suggesting the existence of an additional form of interaction. The function of liganded Hb, on the other hand, remained unchanged in the presence of IHP in excess.

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.

Analysis of Hemodynamic Changes and Bold Signals of Sickle Cell Disease Patients during Desaturation

Introduction: Using near-infrared spectroscopy (NIRS), previous studies have shown that sickle-cell disease (SCD) patients have low cerebral oxygen saturation values [1]. Moreover, the hemoglobin S in sickle cell disease has impaired oxygen carrying capacity [2]. Here, we propose to investigate the effect of induced desaturation on the SCD brain. In particular, we analyzed the falling functional magnetic resonance imaging (fMRI) response during hypoxia, which results from local concentration changes in paramagnetic deoxy-hemoglobin (DHB). Moreover, we also explore the near-infrared spectroscopy (NIRS) changes in oxygenated and deoxygenated hemoglobin from the human prefrontal cortex.Methods: All patients were recruited through informed consent or assent; this study obtained approval from the IRB at Children's hospital Los Angeles. Exclusion criteria included pregnancy, previous stroke, and acute chest pain and crisis hospitalization within one month. Eight patients with SCD and 20 healthy age and ethnicity-matched controls (CTL) were studied. All patients wore oxygen masks in order to control the oxygen level during the experiment in the fMRI. During the scan, the subjects breathed 1) room air for 50 s, 2) 100% N2 for 5 breaths and 3) room air for 6 minutes. The typical length of hypoxia is 5 breaths, but for patients with irregular breathing patterns the maximum duration is 20 seconds. NIRS was used to measure the hemoglobin (OHB, DHB and THB) and a tissue oxygen index (TOI), from the human prefrontal cortex. Differences oh hemoglobin measure, between the normoxia and hypoxia states, were computed and called ΔOHB, ΔDHB and ΔTOI. During resting-state fMRI scanning, subjects were instructed to close their eyes, keep still as much as possible and not to think of anything systematically. FMRI images were acquired during 8 minutes and a total of 240 volumes were collected. Imaging data was first preprocessed with the FMRIB Software Library (FSL), using standard spatial preprocessing steps: Images were (1) slice-time corrected, (2) realigned to remove physiological motion and co-registered to the MNI template space. The percent signal change from the fMRI, % BOLD, was computed based on an average signal in the GM mask.Results: 9 SCD patients (age=23±8) and 20 CTL subjects (age=21±1) were recruited. Fig 1 presents the relationship between %BOLD versus ΔOHB and % BOLD versus ΔDHB, respectively. Due to the lower oxygen affinity of hemoglobin S [1], the decrease in % BOLD (-0.093 ± 0.034) and the ΔOHB (-1.425 ± 0.588) in SCD patients are larger than the decrease in CTL individuals (% BOLD: -0.050 ± 0.021 and ΔOHB: -0.877 ± 0.539); the increase in ΔDHB (1.661 ± 0.805) in SCD patients is also greater than in the control group (ΔDHB: 0.959 ± 0.505), as shown on Figure 2. Based on the THB signal, we can detect hyperemia for most of the subjects during desaturation. Moreover, for some of the SCD patients, the reactivity is slower even at the end of the hypoxic state. We observe a slightly lower increase of ΔTHB for SCD patients, but this was found to be insignificant. This may be the result of a combination of the CBF variability in the SCD group, and of the small sample size. In our study, a decrease of ΔTOI was observed during hypoxia in both groups. The ΔTOI amplitude of the SCD group (-0.080 ± 0.023) is higher than that of the CTL group (-0.047 ± 0.024), which may be due to the SCD brain's diminished physiological adaptive capability to a hypoxic state.Conclusion: Based on a conjoint acquisition of fMRI and NIRS, this study is the first investigation of the effect of the desaturation for SCD patients. As expected, based on these preliminary results, the SCD patients are more affected by hypoxic conditions, such as a larger changes of ΔOHB, ΔDHB, ΔTOI and % BOLD and a slower reactivity, likely due to anemia, high CBF and CBV, and decreased vessel elasticity.†=equal first author contribution

Functional characterisation of the haemoglobins of the migratory notothenioid fish Dissostichus eleginoides

This study addresses the primary structure, the oxygen-binding properties and the CO-rebinding kinetics of the haemoglobins of the Patagonian toothfish Dissostichus eleginoides. D. eleginoides belongs to the family Nototheniidae, the most diversified of the suborder Notothenioidei, mostly exhibiting an Antarctic distribution. Some of its features are typical of Antarctic species, some are not. For instance, D. eleginoides appears not to have functional antifreeze glycoproteins (consistent with its non-Antarctic distribution). In contrast, it has a major and a minor haemoglobin (similar to many Antarctic notothenioids), and their very low oxygen affinity does not follow the trend of other non-Antarctic notothenioids and appears typical of cold-adapted species. Moreover, the amino-acid sequence reveals high identity with the globins of Antarctic notothenioids, arguing in favour of a common origin within notothenioids, and indicates that the primary structure of the major and minor haemoglobins has undergone modifications only to a limited extent. The ligand-rebinding kinetics of the major haemoglobin of D. eleginoides indicate a strong stabilisation of the quaternary T state at lower pH values.