Hb Molecule Research Articles

Preliminary Study on Polymerization between Hemoglobin and Enzymes during the Preparation of PolyHb-SOD-CAT-CA.

The objective of this study was to explore the influence of different factors on the aggregation effect on hemoglobin (Hb) and enzymes during the preparation of Polyhemoglobin-Superoxide dismutase-Catalase-Carbonic anhydrase (PolyHb-SOD-CAT-CA). Several factors including temperatures, pH values, Glutaraldehyde (GDA) amounts and enzymes amounts were investigated systematically to study their effects on the enzymes recoveries and polymerization rates including the Superoxide dismutase (SOD), Catalase (CAT) and Carbonic anhydrase (CA), as well as their effects on the molecular weight distribution of PolyHb-SOD-CAT-CA. Then the oxygen affinity and methemoglobin (MetHb) contents of obtained PolyHb-SOD-CAT-CA were measured to evaluate the effects of enzyme crosslinking on the properties of Polyhemoglobin (PolyHb) moieties in the molecular structure of obtained PolyHb-SOD-CAT-CA conjugate. The results showed that the enzyme recoveries and polymerization rates could be decreased with the temperatures increasing and could be generally kept stable in the physiological pH conditions, but presented only slight changes among the investigated enzyme amounts ranges. Although the GDA concentration increasing could promote the enzyme polymerization rates, the enzyme recoveries decreased in whole. The polymerization rate and molecular size of PolyHb-SOD-CAT-CA conjugate increased with the elevation of temperature and the concentration of GDA. Lastly, the P50 values, Hill coefficients, and MetHb contents of PolyHb-SOD-CAT-CA conjugate with different enzyme crosslinking degrees exhibited no obvious differences with each other. In conclusion, the polymerization reactions between enzymes and Hb molecules could be remarkably affected by temperatures, pH values, and GDA amounts, and the enzyme crosslinking presented no obvious effects on the Hb properties, especially about the oxygen affinity and oxidation degrees.

The Preliminary Study on Preparation Technology of PolyHb-SOD-CAT-CA - The Effects of Different Extractants.

To explore the alternative to extractants for the preparation technology of polyHb-SOD-CAT-CA derived both from bovine blood and human cord blood, compared to the toluene currently used in the existing studies, several organic solvents, including n-hexane and ethyl ether, were investigated during the polyHb-SOC-CAT-CA preparation process. During the technological process, the effects of investigated organic extractants on the properties and stability of hemoglobin and enzymes, including SOD, CAT and CA, were investigated by monitoring the property indexes of macromolecules, including Hb concentrations, MetHb contents, molecular weight distribution, oxygen affinity of Hb, enzymes activities, and so on. Among the investigated experimental groups, the results, including the Hb recoveries, MetHb contents, oxygen affinity, the molecular weight distribution of obtained complex, and enzyme activity, presented to be the best in n-hexane groups, followed by those in toluene groups, and these results in ether groups showed to be the worst. Meanwhile, during the preparation process, both for bovine and human cord derivatives, the changing of properties indexes of Hb and enzymes investigated demonstrated similar declining trends on the whole, and the oxygen-carrying properties and enzyme activities showed to be in the effective range. Among the investigated organic extractants, both for preparation of bovine and human cord-derived polyHb-SOD-CAT-CA complex, n-hexane indicated to have much less negative influence on the properties and stability of Hb and enzyme molecules, including SOD, CAT, and CA. Moreover, the obtained human cord blood polyHb-SOD-CAT-CA also showed effective oxygen-carrying properties and enzyme activities, suggesting the promising application prospects of polyHb-SOD-CAT-CA and the new generation HBOC products in the future.

Studies on the interaction of 2,4-dibromophenol with human hemoglobin using multi-spectroscopic, molecular docking and molecular dynamics techniques

2,4-Dibromophenol (DBP) has several industrial applications, including as a wood preservative and flame retardant. This study investigated the interaction between DBP and human hemoglobin (Hb) using spectroscopic, molecular docking and molecular dynamic techniques. The UV-visible spectra showed ground-state complex formation between DBP and Hb. Fluorescence studies revealed that DBP binding caused significant quenching of Hb fluorescence by the static quenching mechanism. The binding of DBP to Hb is a spontaneous process that involves van der Waals forces and hydrogen bonds. There is one DBP binding site on each Hb molecule that is located at the α1β2 interface of Hb. DBP binding did not alter the microenvironment of tyrosine and tryptophan residues in Hb. Circular dichroism studies revealed that DBP increased the α-helical content of Hb. The intrinsic esterase activity of Hb was inhibited by DBP in a concentration-dependent manner. Molecular docking showed that DBP binds to Hb via hydrogen bonds, hydrophobic, van der Waals and π – π interactions. Molecular dynamics simulation confirmed that the Hb-DBP complex is stable. Overall, the results of this study clearly show that DBP induces structural changes and interferes with the function of Hb. This can have important implications for human health. Communicated by Ramaswamy H. Sarma

Exploring the structural stability of hemoglobin in DBU-based ionic liquids: Insights from spectroscopic investigations

Globular proteins like heme proteins have garnered significant attention owing to their multiple applications in biochemical as well as biophysical processes and are the major functionalities in various pharmaceuticals and health care products. Hemoglobin (Hb), is the widely studied protein in existence and is the most familiar constituent of vertebrate erythrocytes. Protein solubility and stability in aqueous solutions is a major concern to develop therapeutic protein products as the protein lose its activity under unfolded state. Academic and industrial communities have shown great interest in the selection of an apt solvent for biomolecules in suitable biocatalytic applications. Ionic liquids (ILs) have emerged as a suitable cosolvent for the protein molecules by virtue of its high thermal as well as chemical stabilities. Each IL-Protein interaction is specific and generalizing the behaviour is tedious because the same IL can act as both stabilizer and denaturant depending on the external parameters and IL concentration as well. Here in this study, we have synthesized 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) based ILs derived from heterocyclic bases like 1,2,4- triazole, imidazole and morpholine. DBU based ILs are least explored in biomolecule applications but are known for their high thermal stabilities. The interaction of these ILs with hemoglobin was analysed using various spectroscopic techniques including UV–Visible spectroscopy, steady-state fluorescence, Fluorescence Time-Correlated Single Photon Counting (TCSPC), and Dynamic Light Scattering (DLS) to gain better insight into the effects of these ionic liquids on Hb. These results revealed that all the ILs used here are showing Hb friendly behaviour at lower concentrations as it retains the absorbance and fluorescence behaviour of the protein molecule. DLS data explains the solubilization of the Hb molecule, as the ‘z’ average values decreased with periodic addition of the ILs to the system. Quantitative evaluation of the Hb-IL interactions through Benesi-Hildebrand equation suggested that the possibility of complex formations are minimal and this further substantiate that these ILs can act as better cosolvents for Hb in various biochemical reactions.

Water State in Hemoglobin Solutions: Microwave Dielectric Spectroscopy Study

In this review, we discuss the dielectric relaxation of water in the Hemoglobin (Hb) aqueous solutions in different states (MetHb, OxyHb, and DeoxyHb)). The interpretation of the results was performed by the 3D trajectory approach, which considers dielectric parameters of water relaxation and protein concentration. It has been shown that the interaction of amino acid dipole residues, located on the surface of a protein macromolecule, with water dipoles determines the protein hydration. In addition, ions of the buffer, in which proteins are dissolved, are also hydrated. The transition from a dipole-ion interaction of water molecules to a dipole-dipole interaction of water with an increase in protein concentration is observed. We proposed a new approach to calculate hemoglobin hydration shells. The theoretical model defines the change in the ratio between the content of free and bound water molecules (BWM) as a function of MetHb concentration in ion-free and ion-containing aqueous solutions. It considers the number of positive and negative charges at the surface of the protein molecule, the number of BWMs in its hydration shells, and the partition of water molecules bound to MetHb and the inorganic ions. The theoretical evaluation of the ratio of free-to-bound water reveals that, in the absence of ions, MetHb binds about 1400 water molecules, which is in good agreement with experimental data. At MetHb concentrations close to physiological, Hb is dominating in binding water over the inorganic ions, and the amount of BWM remains at the level of ~20% of total cytosol water as the concentration of Hb increases above 15 g/dL. These results suggest that in the physiological concentration diapason (29 - 35g/dL), molecules of MetHb are so close together that their hydration shells interact and shrink from four to one layer of water molecules. In contrast, Hb molecules aggregate to neutralize their surface charges mutually.

Development of biomaterial-based oxygen transportation vehicles for circulation within blood

BackgroundSeveral blood replacements have been developed because of issues with blood collection and storage, increased expenditures, and a global lack of reserves. Studies on artificial blood primarily concentrate on the research and synthesis of molecules that transport oxygen. The primary objective of the current work was to create a supporting, oxygen-carrying biological material that can replace blood and be used to save lives when blood is not available. MethodsThe work started with the synthesis of superparamagnetic iron oxide nanoparticles (SPIONS), and two distinct procedures were applied to ensure binding to hemoglobin (Hb) molecules. The surface modifications of the particles were carried out gradually by 3-amino propyl trimethoxy silane (3APGASPIONS) and tartaric acid (TASPIONS). Analysis methods used to characterize the particles include Fourier transform infrared spectrophotometer (FT-IR), elemental analysis (EDX), scanning electron microscope (SEM), X-ray diffractometry (XRD), and high contrast transmission electron microscopy (C-TEM). The characterization studies of SPIONS which bind Hb molecules to the surface were repeated and their effects on endothelial cell culture were examined to determine their toxic effects in vitro. Cell proliferation, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activity changes were assessed in the evaluation of the possible toxic effects of the cells against the treated particles in the prepared cell lines. Blood substitute solutions synthesized by two different methods were stored on the shelf for about 1 month and the results were compared with fresh Hb solutions. Results and ConclusionThe CV and UV analysis results obtained from artificial Hb substitutes revealed that the structural integrity of Hb molecules did not change, the oxygen transport capabilities of biomolecules were preserved, and this state remained stable for a long time. Furthermore, it was determined that nanocomplexes synthesized by the two methods caused different cellular proliferation and there was a more limited increase in cellular enzyme activities (SOD, CAT, GPx) of Hb-bound particle solutions when compared to pure SPIONS.

Influence of Voxelotor-hemoglobin complexes in the estimation of hemoglobin S levels by the current standard of care laboratory evaluation techniques.

Sickle cell disease is an inherited disorder characterized by the presence of sickle hemoglobin (HbS). The process of Hb molecule polymerization is a pivotal step in the sickling process. Voxelotor, a recently approved novel therapeutic agent, is known to interfere with polymerization. We aim to study the impact of Voxelotor on Hb variants analysis using high performance liquid chromatography (HPLC). We are reporting the impact of Voxelotor on Hb variants analysis using HPLC after an informed consent and medical research committee approval. Data was collected from eight patients who are enrolled in the GBT440-034OL study using electronic medical records, to evaluate the Hb levels, hemolytic markers and the clinical response. Our patients were well-balanced for gender, with a mean age of 31.1 years (19-50). Six patients showed a significant improvement in the Hb level, with reduced reticulocytes, bilirubin, LDH and an improved clinical outcome. Interestingly, these patients showed the appearance of a split band of Hb S and D on HPLC impacting significantly on HbS level. Two patients did not show any improvement on laboratory parameters, and no changes on their HPLC analysis. We report here eight patients on Voxelotor therapy, six of which showed improved hemolytic markers and anemia and demonstrated the appearance of HbD peak on the HPLC chromatogram. Therefore, the absence of HbD on HPLC or other laboratory methods for estimating HbS in patients on Voxelotor therapy, gives the clinician a possible hint regarding the patient's compliance with the drug.

ZIF-8 metal organic framework nanoparticle loaded with tense quaternary state polymerized bovine hemoglobin: potential red blood cell substitute with antioxidant properties.

Due to several limitations associated with blood transfusion, such as the relatively short shelf life of stored blood, low risk of developing acute immune hemolytic reactions and graft-versus-host disease, many strategies have been developed to synthesize hemoglobin-based oxygen carriers (HBOCs) as universal red blood cell (RBC) substitutes. Recently, zeolite imidazole framework-8 (ZIF-8), a metal-organic framework, has attracted considerable attention as a protective scaffold for encapsulation of hemoglobin (Hb). Despite the exceptional thermal and chemical stability of ZIF-8, the major impediments to implementing ZIF-8 for Hb encapsulation are the structural distortions associated with loading large quantities of Hb in the scaffold as the Hb molecule has a larger hydrodynamic diameter than the pore size of ZIF-8. Therefore to reduce the structural distortion caused by Hb encapsulation, we established and optimized a continuous-injection method to synthesize nanoparticle (NP) encapsulated polymerized bovine Hb (PolybHb) using ZIF-8 precursors (ZIF-8P-PolybHb NPs). The synthesis method was further modified by adding EDTA as a chelating agent, which reduced the ZIF-8P-PolybHb NP size to <300 nm. ZIF-8P-PolybHb NPs exhibited lower oxygen affinity (36.4 ± 3.2 mm Hg) compared to unmodified bovine Hb, but was similar in magnitude to unencapsulated PolybHb. The use of the chemical cross-linker glutaraldehyde to polymerize bovine Hb resulted in the low Hill coefficient of PolybHb, indicating loss of Hb's oxygen binding cooperativity, which could be a limitation when using PolybHb as an oxygen carrier for encapsulation inside the ZIF-8 matrix. ZIF-8P-PolybHb NPs exhibited slower oxygen offloading kinetics compared to unencapsulated PolybHb, demonstrating successful encapsulation of PolybHb. ZIF-8P-PolybHb NPs also exhibited favorable antioxidant properties when exposed to H2O2. Incorporation of PolybHb into the ZIF-8 scaffold resulted in reduced cytotoxicity towards human umbilical vein endothelial cells compared to unloaded ZIF-8 NPs and ZIF-8 NPs loaded with bovine Hb. We envisage that such a monodisperse and biocompatible HBOC with low oxygen affinity and antioxidant properties may broaden its use as an RBC substitute.

Haematopoietic Stem Cell Transplantation in Thalassaemia Major: A Narrative Review

Thalassaemia constitutes an especially prevalent human monogenic illness caused by a lack of synthesis of the α- or β-globin chains. The clinical impact of β-thalassaemia is worse since it consists of the same pair gene configuration, thalassaemia major, causing significant health discouragement and loss of life due to life threateningly insufficient haemoglobin (Hb) levels. Only a few nations have successfully reduced the prevalence of β-thalassaemia major, even though comprehensive screening, group counselling, pre-natal detection and public education can all be used. Since over ½ century ago, the fundamental elements of treatment for thalassaemia major have been iron chelation and hypertransfusion. The globin chain that makes up the adult Hb molecule is missing or synthesised at a reduced rate in β-thalassemia. The aberrant buildup of the α-globin chain and faulty formation of red blood cells (RBCs) leading to RBC haemolysis are the outcomes of this genetic abnormality. Since allogeneic haematopoietic stem cell transplantation (Allo-HSCT) has been a well-established gene replacement therapy for individuals with thalassaemia major for several years, it has had very successful outcomes for patients with access to it. Over the past 20 years, the consequences for more susceptible patients have also steadily improved, leading to 80%–90% longer-term life expectancy amongst this group of patients. However, providing Allo-HSCT as a treatment for these patients globally presents numerous difficulties. Replacing genes in autologous HSCs employing viral vectors has been possible in recent years.

Hemoglobin is an oxygen-dependent glutathione buffer adapting the intracellular reduced glutathione levels to oxygen availability

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown.This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.

Изменение конформации и распределения гемоглобина при изменении объема эритроцита

The intracellular space of red blood cells (RBC) includes 40% hemoglobin (Hb) molecules and 60% being water molecules. When the intracellular volume changes, which is possible when erythrocyte cells pass through the blood stream, the possibility of redistribution of intracellular components of the cell due to its volume changes is assumed. By methods of optical spectroscopy (non-invasive methods of infrared spectroscopy, Raman-spectroscopy, laser interference microscopy) changes of morphology, conformation and redistribution of Hb have been revealed in the human erythrocyte due to an increase in the ratio of [Na+]in and [K+]in when Na+/K+-ATPase is blocked in the cell. The decrease of activity of Na+/K+-ATPase by a ouabaine (3 mM) has been found to lead not only to an increase in [Na+]in cell but also to an increase in positive charge on the cytoplasmic surface of the RBC membrane. In these conditions, changes in the conformation of both the heme and globine parts of the cytoplasmic Hb have been identified. It has been shown that cell depolarization, together with cell volume change, leads to a decrease in packing density of Hb molecule, which may be related to sorption of intracellular Na+ (or Ca2+) c Hb, as well as increasing the amount of water molecules in the cell and redistributin Hb in the cell. These processes can lead to a change in the conformation of Hb, as well as to a redistribution and alteration of the conformation of the cytoplasmic Hb.

On the Mechanism of Selective Chemical Exchange of Bacteriopheophytins in the Reaction Centers of Rhodobacter sphaeroides R-26.

To elucidate the mechanism of site-selective chemical replacement of chromophores in the reaction centers (RCs) of photosynthetic bacteria by external pigments, we investigated how the efficiency of incorporation of plant pheophytin a (Pheo) into the binding sites for bacteriopheophytin a molecules (BPheo) in the isolated Rhodobacter sphaeroides R-26 RCs depended on the incubation medium temperature, Pheo aggregation state, and the presence of organic solvent (acetone). When Pheo was in a form of monomers in free detergent micelles in a water-detergent incubation medium, the degree of selective replacement of photochemically inactive BPheo HB molecules upon incubation of the RC/Pheo mixture at 5°C was ~15%. The exchange efficiency increased to 40% upon incubation at 25°C and reached 100% at the same temperature when 10% acetone was added to the incubation medium. At both 5 and 25°C, the degree of pigment exchange increased approximately twice, when a mixture of Pheo monomers and dimers in the presence of 10% acetone was used as the incubation medium. The removal of acetone from this medium with the preservation of pigment forms led to a significant decrease in the efficiency of Pheo incorporation. The effect of acetone on the pigment exchange was also observed at an elevated incubation temperature (43.5°C), when functionally active BPheo HA molecules were partially replaced. The results are discussed in terms of the mechanism according to which (i) the temperature-dependent internal movements of the RC protein facilitate the release of the BPheo molecule from the binding site with simultaneous insertion of the Pheo molecule into the same site in a coupled process, (ii) the role of temperature largely depends on the steric accessibility of binding pockets in the RC protein, (iii) the incorporation of Pheo occurs from a pool of monomeric molecules included in the RC-detergent micelles, and (iv) the presence of acetone in the incubation medium facilitates the exchange of Pheo monomers between micelles in the solution and the detergent belt of the RC complex.

Binding of Quercetin to Hemoglobin Reduced Hemin Release and Lipid Oxidation.

The interactions between quercetin and bovine (or human) hemoglobin (Hb) were systematically investigated by fluorescence, UV-vis absorption spectroscopy, and molecular docking to demonstrate the structural mechanism by which quercetin affected the Hb redox state and stability. Quercetin could interact with the central cavity of the Hb molecule with one binding site to generate an Hb-quercetin complex, and the hydrophobic interaction played an important role in the formation of the complex. The binding constant for the Hb-quercetin complex at 298 K was observed to be 1.25 × 104 M-1. In addition, quercetin effectively inhibited Hb-induced lipid oxidation in liposomes or washed muscles, which was ascribed to the conversion to oxy-Hb and decreased hemin dissociation from met-Hb. Consistent with its lower abilities to bind Hb and scavenge free radicals, rutin (i.e., quercetin-3-rhamnosylglucsoside) did not significantly influence the redox state of Hb nor reduce hemin release from Hb, and subsequently, it less effectively inhibited Hb-induced lipid oxidation than quercetin. Altogether, the results herein provide novel insights into the antioxidant mechanism for quercetin and are beneficial to the application of natural quercetin in Hb-containing foods.

Spectrum of β-Thalassemia and Other Hemoglobinopathies in the Saurashtra Region of Gujarat, India: Analysis of a Large Population Screening Program

Hemoglobinopathies are common genetic disorders of the hemoglobin (Hb) molecule. Globally, 7.0% of the population are carriers of thalassemia with 300,000–400,000 affected births each year. There are >40 million carriers of β-thalassemia (β-thal) in India with 10,000–12,000 affected births every year. This makes control programs crucial in this vast and diverse country. The present study was undertaken to find out the burden of hemoglobinopathies, and in particular, the prevalence of β-thal carriers in the population of Saurashtra region of Gujarat in Western India. A total of 16,780 individuals, including school and college students, were screened. Complete blood counts (CBCs) and high performance liquid chromatography (HPLC) analysis were performed. We detected 1891 (11.26%) individuals with different hemoglobinopathies, of whom 758 (4.52%) were diagnosed to carry β-thal trait, 104 (0.62%) carried Hb D-Punjab (HBB: c.364G>C) trait, 61 (0.36%) carried sickle cell trait, 32 (0.19%) carried δβ-thal trait/HPFH (hereditary persistence of fetal Hb) trait, and other hemoglobinopathies were identified in smaller numbers (0.15%). We encountered 27 individuals with mean corpuscular Hb (MCH) <27.0 pg and mean corpuscular volume (MCV) <80.0 fL levels, who had borderline Hb A2 levels (3.2–3.5%). Twenty castes showed the presence of β-thal or other hemoglobinopathies. A high prevalence of β-thal was found in the Sindhis (11.67%), Lohanas (9.71%), Brahmins (6.31%), Bharvads (6.94%), Harijans (7.57%) and Vankars (7.77%). All the heterozygotes were given appropriate counseling. A multi pronged approach, including screening of high school and college students, needs to be considered for this vast and ethnically diverse country to reduce the burden of hemoglobinopathies.

Angelica archangelica and Ginkgo biloba Extracts Recover Functional Blood Hemoglobin Derivatives in Rabbits Exposed to High Altitude.

Shortage of oxygen is a common condition for residents of high-altitude (HA) areas. In mammals, hemoglobin (Hb) has four derivatives: oxyhemoglobin (Hb-O2), carboxyhemoglobin (Hb-CO), sulfhemoglobin (Hb-S), and methemoglobin (Met-Hb). In HA areas, aberrant physiological performance of blood hemoglobin is well-established. The study aimed to investigate the influence of 30 days of HA residence on rabbits' total Hb, Hb derivatives, Hb autooxidation rate, and antioxidant enzymes in comparison to low-altitude control rabbits. Further, the study aimed to investigate the effect of antioxidant-rich Angelica archangelica and/or Ginkgo biloba extracts on the same parameters in HA-resident rabbits. Rabbits subjected to 30 days of HA residence were compared to low-altitude control rabbits. HA-residence rabbits were then orally administered 0.11 g/kg b.wt. of Angelica archangelica and/or Ginkgo biloba extract for 14 days. Hb derivatives and Hb autooxidation rate were measured spectrophotometrically. Antioxidant enzymes were estimated using specialized kits. Compared to low-altitude rabbits, 30-day HA-residence rabbits showed a noticeable increase (p<0.05) in Hb-O2 and Hb-CO concentration. In addition, Met-Hb concentration, autooxidation rate of Hb molecules, and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) exhibited a remarkable increase in HA-residence rabbits (p<0.01), reflective of rapid ROS generation. In HA-residence rabbits, both individual and combined treatment with antioxidant-rich extracts for 14 days resulted in recovery to near-normal functional levels of Hb-O2 and Met-Hb, Hb autooxidation rate, and activities of SOD and GPx, while only combined treatment led to Hb-O2 recovery. The findings suggest that functional Hb levels may be recovered by oral administration of A. archangelica, G. biloba, or combined treatments. In conclusion, oxidative stress due to living in HA areas may be avoided by supplementation with natural antioxidants.

Greek National Prevention Program for Thalassemia and Other Hemoglobinopathies and Contemporary Bioethical Reflections. The Experience of Northern Greece

Hemoglobinopathies constitute the most frequent monogenic disorders worldwide and are caused by gene mutations that affect the synthesis of the normal hemoglobin chains or contribute to structural changes of the Hb molecule. In Greece hemoglobinopathies are the most frequent genetic diseases and the Greek National Prevention Program for Thalassemia and other Hemoglobinopathies is the most typical international example of a voluntary prevention screening program. During a 20-year study period, gene interactions in 437 pregnancies were related with risk of β-thal major, sickle cell disease and Hb Bart’s hydrops fetalis. 437 prenatal diagnoses were performed and (22.2%, N=97) fetuses were homozygotes or compound heterozygotes for clinically significant mutations. The couples were informed of all the currently available therapeutic approaches for β-TM or sickle cell disease. The decision for the termination or continuation of the pregnancy was left to parental choice and the termination of the pregnancy was generally accepted and 91 from 97 affected pregnancies were terminated. The “success” of a prevention program depends on the commitment and the will of the governments, the consolidation of strong educational campaigns, the continuing public education and health professional awareness, the existence of reliable laboratories. Greek National Bioethics &amp; Techno ethics Committee gives a framework about the bioethical questions arising as well as the “Bio laws” in the Greek constitution. In our constitution the availability of prenatal diagnosis, the social bioethical framework as expressed by the National Bioethics committee, the contemporary role of religion in public life (Greek Orthodox Church less influential) and the existing legal frame of the termination of pregnancy in cases of severe hemoglobinopathies contributed to the success of the Prevention programme.

Examination of the primary fraction of hemoglobin in individuals having sickle cell disease using high-performance liquid chromatography

Background: Hemoglobinopathies refer to a range of diseases, in which the hemoglobin (Hb) molecule’s synthesis or structure is aberrant. It is passed down through the generations (inherited). Hb C illness, Hb S-C disease, sickle cell anemia, and thalassemia are all part of this group. Hemoglobinopathies are the most frequent genetically inherited red blood cell condition on the planet. Hb variant screening and precise identification have become more crucial in the antenatal diagnosis and prevention of Hb diseases. Aims and Objectives: Hb analysis, cation exchange high-performance liquid chromatography (HPLC) has emerged as the method of choice. This research was brought up to diagnose numerous hemoglobinopathies and also to measure distinct Hb fractions. Materials and Methods: Four hundred and seventy participants were examined for hemoglobinopathies in the investigation. Different fractions of Hb were compared to patients with normal Hb after diagnosis. Results: Normal HPLC, SCA (Hb SS), sickle cell trait (SCT), and -thalassemia trait were discovered in 110 (23.4%), 130 (27.6%), 210 (44.6%), and 20 (4.2%) individuals, respectively, out of 470 patients. Rare hemoglobinopathies such as tenacious fetal Hb (PFH), Hb E, Hb D-Punjab, and Hb Q India accounted for 15 (3.1%) of the sufferers. Patients with SCA had a mean percent of Hb S, Hb F, Hb A, and Hb A2 of 81.6±74.6, 22.9±13.9, 3.1±6.1, and 2.5±1.02, respectively. Hb F was 22.9±13.9% on average, which was much higher than normal and SCT. A high HbA2 level (&gt;3.5%) indicated the existence of the β-thalassemia trait. Conclusion: In the included section of the population, SCD is the most common kind of hemoglobinopathy, both homozygous and heterozygous. Hb F concentrations are observed to be elevated in SCA patients, and it is thought to be a key element in protecting patients from problems. Raised Hb A2, which indicates the presence of the β-thalassemia trait, was also discovered in this area, and it is suspected when the value is &gt;3.7%. Cation exchange HPLC is ideal for routine hemoglobinopathies research due to its high resolution, short test time, and precise quantification.

Microfluidic Microcirculation Mimetic as a Tool for the Study of Rheological Characteristics of Red Blood Cells in Patients with Sickle Cell Anemia

Sickle cell disorder (SCD) is a multisystem disease with heterogeneous phenotypes. Although all patients have the mutated hemoglobin (Hb) in the SS phenotype, the severity and frequency of complications are variable. When exposed to low oxygen tension, the Hb molecule becomes dense and forms tactoids, which lead to the peculiar sickled shapes of the affected red blood cells, giving the disorder its name. This sickle cell morphology is responsible for the profound and widespread pathologies associated with this disorder, such as vaso-occlusive crisis (VOC). How much of the clinical manifestation is due to sickled erythrocytes and what is due to the relative contributions of other elements in the blood, especially in the microcapillary circulation, is usually not visualized and quantified for each patient during clinical management. Here, we used a microfluidic microcirculation mimetic (MMM), which has 187 capillary-like constrictions, to impose deformations on erythrocytes of 25 SCD patients, visualizing and characterizing the morpho-rheological properties of the cells in normoxic, hypoxic (using sodium meta-bisulfite) and treatment conditions (using hydroxyurea). The MMM enabled a patient-specific quantification of shape descriptors (circularity and roundness) and transit time through the capillary constrictions, which are readouts for morpho-rheological properties implicated in VOC. Transit times varied significantly (p &lt; 0.001) between patients. Our results demonstrate the feasibility of microfluidics-based monitoring of individual patients for personalized care in the context of SCD complications such as VOC, even in resource-constrained settings.

Hemoglobin bio-adhesive nanoparticles as a colon-specific delivery system for sustained release of 5-aminosalicylic acid in the effective treatment of inflammatory bowel disease

A colonic drug delivery system was developed to specifically deliver 5-aminosalicylic acid (5-ASA) to the inflamed site by conjugating with hemoglobin nanoparticles (HbNPs). The 5-ASA-HbNPs (eight 5-ASA molecules per Hb molecule) with the size of 220 nm and zeta potential of −14.6 mV is a tailored nanoparticle able to pass through the mucus layer. The 5-ASA-HbNPs do not undergo chemical and enzymatic hydrolysis in the simulated gastrointestinal fluids over 6 h. Significantly higher cellular uptakes and prolonged release was seen for the 5-ASA-HbNPs in Caco-2 cells, compared to free 5-ASA over 72 h. In addition, 5-ASA-HbNPs revealed similar therapeutic effectiveness with free 5-ASA against tumor necrosis factor and showed less inhibitory concentration (IC50) for myeloperoxidase enzyme activity. In vivo imaging of mouse demonstrated the localization of drug in the descending colon after oral administration and about 15% of the administered dose was recovered as 5-ASA from urine in 6 h. The use of these nanoparticles with the mucus adhesion properties and permeability to intestinal epithelial cells can be a good candidate with potential application in the colonic drug delivery field.

Calorimetric Characterisation of the Binding Reaction Between Human Ferric Haemoglobins and Haptoglobin to Develop a Drug for Removal of Cell-Free Haemoglobin.

High levels of cell-free haemoglobin (Hb) may occur in plasma as a consequence of e.g., pathological haemolysis or blood transfusion. These Hb molecules can be removed from blood circulation by forming a complex with the acute-phase protein haptoglobin (Hp) and thereby can also the intrinsic toxicity of free Hb be limited. In this study it is shown that ferric HbA, HbF, HbE and HbS, respectively, all bind firmly to Hp at 25°C. By using isothermal titration calorimetry (ITC), it is demonstrated that ferric HbF has higher affinity to Hp (Ka=2.79±0.29 ×109 M-1) compared with HbA and HbS (1.91±0.24 ×109 M-1) and 1.41±0.34 ×109M-1 for HbA and HbS, respectively. In addition, the affinity constant for HbE is slightly lower than the other haemoglobins (0.47±0.40 ×109M-1). Since Hp shows a general and high affinity to all Hb variants tested, it can be concluded that Hp may be useful as a therapeutic agent for several different haemolytic conditions by intravenous injection.