HbSS Blood Research Articles

Povišena srednja zapremina eritrocita u anemiji srpastih ćelija - jedna priča, previše?

Abstract: Introduction: Sickle cell disease is a hereditary blood disorder characterized by defective hemoglobin. Red cell indices are proposed as potential tools for diagnosing and managing sickle cell disorders. Materials and Methods: This study aimed to assess the utility of red cell indices as screening tools for sickle cell anemia. One hundred consenting adults of both sexes participated. Haematological parameters, including packed cell volume, hemoglobin values, hemoglobin electrphoretic patterns, and red blood cell count, were examined. Mean cell volume (MCV), mean cell hemoglobin concentration (MCHC), and mean cell hemoglobin (MCH) were calculated. Data analysis was performed using GraphPad Prism Software Version 9, with statistical significance set at p < 0.05 (95% confidence interval). Results: Haemoglobin values were significantly lower in Hb SS subjects (5.68 ± 1.7g/dl) compared to Hb AA (11.30 ± 1.5 g/dl) and Hb AS groups (11.03 ± 1.4 g/dl) (F = 32.279; p < 0.00001). The pattern was consistent with PCV and RBC values. Among the red blood cell indices assessed, only MCV showed a significant elevation (95.7 ± 2.4 fl) in the HbSS group compared to other groups (F = 4.165; p = 0.0183). No statistically significant difference was observed in MCHC and MCH values between the three groups (F = 0.5373, p > 0.586 for MCHC; F = 0.607, p > 0.546 for MCH). The prevalence of haemoglobin variants was as follows: HbAA (77%), HbAS (19%), and HbSS (4%). Conclusion: This study highlights significant reductions in haemoglobin values in Hb SS subjects and a notable elevation in MCV values in the Hb SS blood group. Elevated MCV in sickle cell anemia, where red cells are typically microcytic, warrants further investigation for differential diagnosis.

P-099: ANTI-SICKLING STUDY OF THE AQUEOUS EXTRACTS OF TINOSPORA CARDIFOLIA AND TERMINALIA BELLIRICA

Purpose: Introduction: - Sickle cell anaemia also known as drepanocytosis, is a genetic blood related disease in which red blood cells form an cresent (sickle) or abnormal shape. This genetic disease is due to a change in the beta globin chain of haemoglobin glutamic acid (a polar amino acid) is replaced by valine (a non – polar) amino acid. This genetical change reduces the binding of haemoglobin for oxygen. At the low oxygen level the mutant haemoglobin, known as sickle haemoglobin (Hbss). Medicinal plants have been used in Indian folk medicine in the remedy of sickle cell anaemia by inhibiting sickling. This work was therefore aimed at screening the antisickling study of the aqueous extract of stems of T. Cardifolia and fruits of T.bellirica used in the Indian herbal medicine. Objective:- The present study investigates the preliminary screening of secondary metabolites of aqueous extracts of T. Cardifolia(stems) and T.bellirica(fruits) and invitro antisickling test (sickling reversal test). Materials and methods: Standard methods of phytochemical analysis of T. Cardifolia(stems) and T.bellirica(fruits) and sickling reversal test (invitro antisickling test), stems of T. Cardifolia,fruits of T.bellirica and Hbss blood samples etc. Results: Screening of secondary metabolites (bioactive molecules) and sickling reversal test were carried out using standard methods. The phyto-chemical screening indicated the presence of secondary metabolites (bioactive molecules) viz - alkaloids,flavonoids,phenols, saponins, steroids and cardiac glycosides etc. The results of antisickling reversal test that T. cardifolia(stems) and T. bellirica (fruits) exhibited potential antisickling property. Conclusion: The percentage of sickle cells of untreated Hbss was statistically significant higher than treated Hbss. The percentage of sickle cells decreased on the addition of aqueous extract of T. cardifolia stems and T. bellirica fruits on induced Hbss blood, which indicates the reversal of of Hbss blood to normal biconcave shape due to the presence of bioactive molecules, which have sickling reversal (anti-sickling property) and anti haemolytic properties. The authors do not declare any conflict of interest

Synthetic Heparan Sulfate Compounds Attenuate Vascular Complications Associated with Sickle Cell Disease

In sickle cell disease (SCD), a mutation of the β-globin gene leads to abnormal polymerization of hemoglobin, resulting in formation of sickled red blood cells, hemolytic anemia, and vaso-occlusive crisis (VOC). These primary events produce clinical complications and trigger additional multiple pathologies driven by chronic oxidative stress, sterile inflammation, and activation of coagulation.Heparins, highly sulfated forms of heparan sulfate (HS), are a group of polysaccharide compounds with great variance in structure. In addition to their anticoagulant effect, heparins have anti-adhesive and anti-inflammatory properties. These are determined by both sulfation pattern and length of the polysaccharide chain, which influence the ability to bind HMGB-1, histones, and P-selectin (Psel). This heterogeneity together with the short half-life and dosing regimen based on anticoagulant activity, limit the use of heparins as anti-adhesive and anti-inflammatory agents. To overcome these limitations, we previously developed a chemoenzymatic approach to synthesize a structurally defined HS oligosaccharides and demonstrated their ability to reduce sterile inflammation in animal models by binding HMGB-1 and histones. In the present study, we investigated the compound's anti-Psel properties in vitro and in a mouse model of SCD.First, using a Psel inhibition ELISA assay, we determined that a heptadecasaccharide (17-mer) is the minimum polysaccharide chain length required for inhibition of Psel binding to Sialyl Lewis X polyacrylamide. Based on these data, we synthesized three 18-mer compounds with a different sulfation position on each monosaccharide ring (NS2S, NS6S and NS2S6S). To obtain 18-mers with no anticoagulant activity, we omitted 3-O-sulfation of glucosamine, which is important for binding antithrombin III (confirmed by anti-FXa activity assay). In the Psel inhibition ELISA assay, all compounds demonstrated dose dependent (0.1 - 1000 µg/mL) anti-Psel activity comparable to that observed for low molecular weight heparin (LMWH).Psel is a key molecule mediating VOC by promoting formation of multicellular aggregates. Therefore, we evaluated the effect of 18-mers on Psel-mediated platelet/leukocyte aggregates (PLA) formation ex vivo. Leukocytes and platelets isolated from healthy donors were stimulated with PMA (100 nM) for 1 hour or thrombin (5 µg/mL) for 30 minutes, respectively, then incubated with vehicle, 18-mer compounds, or LMWH (0.5, 5, 50 and 500 µg/mL) for 15 minutes. After incubation, cells were combined to allow PLA formation for 15 minutes and analyzed by flow cytometry. At the highest tested 18-mer concentration, all compounds attenuated PLA formation. However only NS2S6S, the most highly sulfated compound, showed significant inhibition at all concentrations. NS2S6S decreased PLA formation to 82.8% 90.8%, 76.3% and 68.3%, lowest to highest concentrations respectively (p<0.01 for all concentrations versus vehicle). LMWH demonstrated significant decreases only at the two highest concentrations (82.1% and 63.8%, p<0.001). The number of circulating PLA was increased in sickle Townes HbSS mice by 6.1-fold (p<0.01) compared to non-sickle Townes HbAA controls. In ex vivo experiments, addition of NS2S6S (1 mg/ml) to the HbSS blood decreased PLA formation to 66.4% (p=0.03) compared to untreated HbSS blood.Finally, we determined the effect of NS2S6S on heme-induced microvascular stasis in Townes HbSS mice. Sickle mice were implanted with a dorsal skinfold chamber to visualize dermal microvessels. PBS or NS2S6S (3 mg/kg, s.c.) were injected 15 min before infusion of heme (1.2 µmol/kg, iv). 0ne, 2, 3 and 4 hours after heme infusion, microvascular stasis was observed in 31.5, 20.5, 18.9 and 14.2% of preselected vessels in PBS treated sickle mice, and NS2S6S treatment reduced that numbers to 10.9, 6.2, 3.1 and 3.2%, respectively (p<0.01 for all time points).In summary, we showed that NS2S6S prevents Psel dependent formation of PLA ex vivo and reduces heme-induced stasis in sickle mice. Together with previously described anti-HMGB1 and anti-histone effects, this compound is a good candidate for multi-modal therapy to mitigate the pathophysiology of SCD. However, like LMWH, NS2S6S has a short half-life which makes prophylactic treatment of SCD patients impractical. Studies to extend the half-life of HS are currently ongoing in our group. DisclosuresXu: Glycan Therapeutics: Current Employment. Belcher: Mitobridge/Astellas: Consultancy, Research Funding; CSL Behring: Research Funding. Vercellotti: CSL Behring: Research Funding; Mitobridge, an Astellas Company: Consultancy, Research Funding. Liu: Glycan Therapeutics: Current Employment.

Invitro Membrane Stabilizing Potentials Of Fractionates Of Ethanolic Extract of Carica Papaya Leaf

Invitro membrane-stabilizing potentials of fractionates of ethanolic extract of Carica Papaya leaf was investigated in this study. The soxhlet extraction method was used to extract the plant, fractionated with 6 different solvents to give 6 different fractions (hexane, ethyl acetate, chloroform, butanol, methanol benzene). Hbss red blood cells samples were obtained from non-crises state sickle cell patients from Eku Baptist hospital Abraka Delta State, Nigeria. These tests involved the use of positive (p-hydroxy benzoic acid 5ug/ml) and negative controls (normal saline) for membrane stability experiments. Hbss blood was treated with 2mg/ml to 10mg/ml in seven groups with leaf fractionates. Data was analyzed using ANOVA test. The results shows that osmotic fragility was reduced by the introduction of the leaf fractionate, with the highest rate of reduction noticed in the hexane 1 fractionate. PHBA reversal rate and osmotic fragility effect was normal at low doses, but as concentration increases, reversal rate and percentage reduction of sickling decreases. It was concluded that Carica papaya leaf extract fractions, just as its crude extracts, have as much osmotic fragility activities, and this is dose-dependent and has no negative effect on tested blood samples as compared with the treatments with PHBA.

Amino Acids Extract from Black Bean Seeds (Phaseolus Vulgaris L.)Inhibit Hemoglobin Polymerizationand Modulate Erythrocyte Membrane Atpases In Sickle Cell Disease (SCD)

Back ground: The basic extracts involved in the formulation of functional foods/nutraceutics must first be the subject of preclinical and clinical trials. The several studies were shown that amino acids extract from black bean seeds (Phaseolus vulgaris L.) have antisickling, antioxidant and anti-haemolitic properties. Objective: The objective of this study is to investigate the effect of amino acids extract from black bean seeds (Phaseolus vulgaris L.) on hemoglobin polymerization and erythrocytes membrane ATPases activities in Sickle Cell Disease (SCD). Method: The ability for the amino acids extract to inhibit sickle cell hemoglobin polymerization and improve the Fe2+/Fe3+ ratio of HbSS blood were assed using spectroscopic method. The activities of three ATPases (Na+/K+; Ca2+; and Mg2+-ATPases) were investigated by the kinetic method following the release of inorganic phosphate (Pi) by ATP taking into account the determination of the total membrane proteins of erythrocytes by Bradford’s method. Result: The lowest potential of inhibition was obtained (39.98±2%) with the amino acids extract at 12 mg/mL corresponding significantly (p&lt;0.05) to the highest potential of inhibition of polymerization (71.16±2%). The greatest Fe2+Fe3+ ratio was obtained (3.94±2%) with the amino acids extract at 12 mg/mL corresponding significantly (p&lt;0.05) to the best rates increase of oxy-hemoglobin (48.73±2%). The red blood cells membranes ATPase activity were estimated and expressed as μmol of Pi/mg of protein/hour x 10-3 there was an increase in the activities of Na+/ K+ and Ca2+-ATPases (255.98±62 and 282.98±36 respectively) at the concentration of 12 mg/mL of extract compared to the negative control (167.36±60 and 236.91±62 respectively). However, for Mg2+-ATPase, there is rather a significant reduction (p&lt;0.05) in this activity (286.71±51 against 146.53±35) obtained in the presence of the amino acid extract at 12 mg/mL). Conclusion: The amino acids extract of black bean seeds (Phaseolus vulgaris L.) at a concentration of 12 mg/mL offered the best inhibition potentials of hemoglobin S polymerization, increase rates of oxyhemoglobin and the best ATPasic activities. Keywords: Amino acid extracts; black bean seeds; sickle cell disease; polymerization; Fe2+/Fe3 + ratio; ATPase activities

Phytochemical Characterization of Three Plants and Their Antisickling Activity in the Management of Sickle Cell Disease

Natural plant products have been used by the population of the south-eastern part of Cote d’Ivoire in the management of sickle cell anemia. This study was aimed at investigating the antisickling activity of the hydro-ethanolic extract of a combination of the leaves of J. secunda, J. gossypiifolia and P. nigrescens. These three plants species were used in the Ivorian traditional herbal medicine. Preliminary phytochemistry was carried out using standard methods. As for the sickling reversal test, the Hb SS blood sickling was induced with 2% sodium metabisulfite. After 120 minutes of incubation, the plant extract was added. Every 30 minutes for a period of 120 minutes, a drop of the prepared solution was observed at (40×) magnification and the percentage of reversion calculated. The phytochemical analysis revealed the presence of Alkaloids, Flavonoids, Polyphenols, Catechic Tannins, Sterols and Polyterpenes. The results of the reversal test showed that the percentage of sickling reversal effect of the combination of the plants (75.00 ± 4.33b,c) was highly superior than the negative control (10.17 ± 0.55d) but was significantly (p J. secunda: 83.50 ± 2.33a; J. gossypiifolia: 78.00 ± 3.67b,c and P. nigrescens: 77.83 ± 2.89b,c) and the positive control (80.66 ± 2.22a,b). From the results, the extracts J. secunda, J. gossypiifolia and P. nigrescens have shown to be therapeutically beneficial to the population. Their use is also justified in the management of sickle cell disease in the south-eastern part of Cote d’Ivoire.

Antisickling Activities of Some Nigerian Medicinal Plants

Sickle cell anaemia (SCA) is a genetic condition caused by defective haemoglobin molecules. Symptoms are often associated with painful crisis often accompanied with vaso-occlusion, severe anaemia, acute chest syndrome, stroke, multiple organ failure, etc. In the pursuit of searching for suitable and affordable anti-sickling herbal remedies, aqueous and ethanolic extracts of ten medicinal plants from relevant previous ethnomedical records, were screened using standard methods involving whole HbSS blood and found to possess varying degrees of anti-sickling activities. For example, Anthocleista vogelii Planch (Gentiaceae) gave 12% inhibitory and 61% reversal activities; Cassia sieberiana DC ripe fruit and pericarp (Leguminoseae) gave 54% inhibitory, 90% reversal activities and 71% inhibitory and 54% reversal activities, Harungana madagascariensis Lam. ex. Poir (Hypericaceae) gave 42% inhibitory and 52% reversal activities and Senna alata (L.) Roxb. (Leguminoseae) gave 19% inhibitory and 84% reversal activities. Thus, the pericarp and whole fruit extracts of Cassia sieberiana pericarp gave the best inhibitory activity (71%) and reversal (90%) activities respectively while S. alata appeared to be a competing candidate for reversal activity (84%). Consequently, an appropriate herbal combination research on the three plant materials to achieve the best inhibitory and reversal activities in a single formulation is in progress.Keywords: Anti-sickling, Cassia sieberiana, Senna alata

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Evaluation of the Amino Acid Profile and Haemoglobin Polymerization Inhibition Potential of Some Nigerian Legumes

The amino acid profile and the effects of the seed extracts of Sphenostylis sternocarpa , Monodora myristica and Mucuna sloanei were studied based on their ability to inhibit haemoglobin polymerization and improve the Fe 2+ /Fe 3+ ratio of sickle cell erythrocytes. The samples were fractionated into crude aqueous extract (CAE), fat-soluble (FAS), butanol-soluble (BUS) and water-soluble (WAS) fractions. The CAEs of the samples ranked highest in amino acid content in the range of S. sternocarpa (7.12 ± 0.00 g/100g)>M. myristica (6.00 ± 0.15 g/100g)>M. sloanei (3.56 ± 0.21 g/100g). The amino acids identified in appreciable quantities in the seed samples included Phe, Leu, Val, Ile, His, Arg, Tyr, Met, among others. The extracts inhibited polymerization to varying degrees with CAE of both S. sternocarpa and M. myristica , as well as the WAS of M. myristica eliciting significantly (p<0.05) high percent inhibition of polymerization when compared with Phe standard. The extracts improved the Fe 2+ /Fe 3+ ratio of HbSS blood from 1.36% for CAE of M. sloanei to 85.04% for CAE of S. sternocarpa ; and from 11.03% for WAS of S. sternocarpa to 36.08% for WAS of M. sloanei . These legumes could, therefore, have immense nutritional and therapeutic importance in the management of sickle cell disease and other related diseases.

Antisickling and toxicological profiles of leaf and stem of Parquetina nigrescens L.

Folk medicine reportedly uses Parquetina nigrescens L. (Asclepiadaceae) as a herbal remedy for the management of sickle cell anemia. This study was carried out to screen the leaves and stem of P. nigrescens for antisickling activity, erythrocyte membrane -stabilizing effects and any end organ toxicity. Percentage Reversal and Inhibition of Sickling parameters were analyzed on pre-sickled HbSS blood cell suspensions using sodium metabisulphite solution as inducer and 5 mg/ml parahydroxybenzoic acid and normal saline as positive and negative controls respectively. Effects of the plant extracts on the erythrocyte were assessed using osmotic fragility and the toxicity profile done via LD50 and Sub-Acute toxicity studies on graded concentrations of extract. Results show that P. nigrescens has appreciable antisickling activity, has no toxic effect when administered at low concentrations and protects the integrity of the erythrocyte membrane as evidenced in the fragiliogram by the reduction in hemolysis of the Hbss cells. Key words: Antisickling activity, membrane integrity, osmotic fragility,Parquetina nigrescens, sickle cell disease.

Anti sickling potential of Aloe vera extract IV: Effects of acidic, basic and neutral metabolites on the gelling and sickling of human HBSS erythrocytes

The effects of acidic, basic and neutral metabolites from A. vera extract on the sickling and gelling time of human HbSS erythrocytes were investigated. The study showed that the various metabolites inhibited sickling in vitro and increased gelling time of HbSS blood samples. Moreover, a significant increase in the number of unsickled erythrocytes was observed for the acidic metabolites. The abilities of the metabolites to revert already sickled erythrocytes to their normal morphology was highest for the acidic metabolites, but non–existent for the neutral metabolites, which appeared to be toxic to the HbSS blood cells. Therefore, it was concluded that the reversal of the sickling phenomenon could be achieved, almost quantitatively, by the consumption of acidic metabolites by sickle cell patients. Keywords: Sickle cell disease, fractionated A. vera extract, gelling time International Journal of Natural and Applied Sciences , 6(2): 155-160, 2010

Anti‐sickling potential of Aloe vera extract

AbstractThe effect of Aloe vera extract on the gelling time of human HbSS erythrocytes was investigated. The results showed that A. vera extract increased the gelling time of HbSS blood and inhibited sickling in vitro. In addition, a linear relationship was found between extract concentration and gelling time, suggesting that A. vera extract may have great potential in the management of sickle cell disease. Copyright © 2008 Society of Chemical Industry

Effects of aqueous extracts of &lt;i&gt;Zanthoxylum macrophylla&lt;/i&gt; roots on membrane stability of human erythrocytes of different genotypes

The effects of aqueous extract of Zanthoxylum macrophylla (formerly Fagara xanthoxyloides) roots on the membrane stabilities of human erythrocyte from HbAA, HbAS and HbSS blood were investigated. The determination of mean corpuscular fragility (MCF), which is the concentration of saline causing 50% haemolysis of the erythrocytes, showed that the aqueous plant extract increased the MCF values of the various erythrocytes. There was no significant difference (p>0.05) between the MCF values of HbAA and HbAS erythrocytes. However, significant difference (p

Effects of aqueous extracts of &lt;i&gt;Garcinia kola&lt;/i&gt; seeds on membrane stability of HbAA, HbAS and HbSS human erythrocytes

The effects of water-extract of the seed of Garcinia kola (Henckel), a popularly consumed seed known as ‘bitter kola' in Nigeria, on the stabilities of erythrocyte membranes from HbAA, HbAS and HbSS blood donors were determined. The median corpuscular fragility (MCF) of erythrocytes from the three genotypes were 3.6 + 0.1, 3.4 + 0.1 and 2.8 + 0.2, expressed in [NaCl] g/l for HbAA, HbAS and HbSS, respectively. The aqueous extract from G. kola seeds stabilized the membranes from HbAA, HbAS and HbSS erythrocytes by 22, 17 and 25% respectively. Phenylalanine, a known anti-sickling agent, stabilized the HbSS membrane by 17.85%. Viscosity studies indicated that the HbSS blood was more viscous and the aqueous extract from G. kola seeds decreased the viscosity of the HbSS blood, producing a statistically different (p

Hydroxyurea-induced oxidative damage of normal and sickle cell hemoglobins in vitro: amelioration by radical scavengers.

Hydroxyurea (HU) induces fetal hemoglobin (Hb F) production in patients with sickle cell anemia. The therapeutic dosage of HU used for Hb F induction often elicits myelosuppression, which becomes its major associated complication. We examined the effect of HU on hemoglobin modulation and the role of radical scavengers on these induced changes. In vitro exposure of human blood to various concentrations of HU at predetermined time intervals induced a progressive dose-dependent oxidation (MetHb formation) of both adult (Hb AA) and sickle (Hb SS) hemoglobins. The oxidative effect of HU on Hb SS was 3 times greater than its effect on Hb AA. Similar but less profound changes were observed in H2O2-treated samples. Hb F was, however, observed to be relatively resistant to HU-induced oxidative damage. A substantial protective effect of Hb by alpha-tocopherol, ascorbic acid, and D-mannitol was observed during pretreatment of Hb AA and Hb SS blood samples. Analyses of the hemoglobins and their globin chain components by high-performance liquid chromatography revealed a considerable protective effect by these free radical scavengers. These results indicate that the HU-induced damage of hemoglobin and their component globin chains can be reduced by radical scavengers.

Effect of Zanthoxylum xanthoxyloides and some substituted benzoic acids on glucose-6-phosphate and 6-phosphogluconate dehydrogenases in hbss red blood cells

The ether fraction of the aqueous extract of the roots of Zanthoxylum xanthoxyloides (antisickling fraction), vanillic acid, parahydroxybenzoic acid and paraflurobenzoic acid possess antisickling inhibitory activity at low con- centrations. Paraflurobenzoic acid was the most active. When the activities of NADP +-linked glucose-6-phosphate and 6-phosphogluconate dehydrogen- ases in washed Hbss blood specimens treated with these agents (6 mg/ml for antisickling fraction and 6 Mg/ml for the acids) were compared with controls in vitro, there were no significant differences in either normal or sickled states. The media were devoid of enzyme activity. These agents neither affect the activities of these enzymes while exhibiting antisickling activity nor disrupt the cell membrane to the extent of causing leakage to the media.

Water Distribution in Blood During Sickling of Erythrocytes

Plasma urea and protein determinations proved suitable for measuring changes in total diffusible water and plasma volume in whole blood. Deoxygenation by saturation with carbon dioxide at 25 degrees C caused no change in plasma urea, but a significant increase in plasma protein concentration was induced with both normal and sickle-cell (HbSS) blood. Thus in HbSS blood there was no binding or trapping of water as a result of sickling and there was a normal influx of water into the cells (Bohr effect) despite the polymerization of the hemoglobin molecules with sickling. Consistent with this observation was the finding that the deoxygenation induced a similar increase in concentration of the plasma cations, sodium plus potassium. HbSS erythrocytes neither lost nor gained water under the more physiologic conditions of deoxygenation with a 95% nitrogen, 5% carbon dioxide gas mixture.

Water distribution in blood during sickling of erythrocytes

Plasma urea and protein determinations proved suitable for measuring changes in total diffusible water and plasma volume in whole blood. Deoxygenation by saturation with carbon dioxide at 25 degrees C caused no change in plasma urea, but a significant increase in plasma protein concentration was induced with both normal and sickle-cell (HbSS) blood. Thus in HbSS blood there was no binding or trapping of water as a result of sickling and there was a normal influx of water into the cells (Bohr effect) despite the polymerization of the hemoglobin molecules with sickling. Consistent with this observation was the finding that the deoxygenation induced a similar increase in concentration of the plasma cations, sodium plus potassium. HbSS erythrocytes neither lost nor gained water under the more physiologic conditions of deoxygenation with a 95% nitrogen, 5% carbon dioxide gas mixture.

Erythrocyte Hb-S concentration. An important factor in the low oxygen affinity of blood in sickle cell anemia.

The blood in sickle cell anemia has a very low oxygen affinity and, although 2,3-diphosphoglycerate (2,3-DPG) is increased, there is doubt as to whether this is the only factor responsible. In this study of 15 patients with sickle cell anemia (Hb SS) no correlation was found between oxygen affinity (P(50) at pH 7.13) and 2,3-DPG in fresh venous blood. Whole populations of Hb SS erythrocytes were therefore separated, by an ultracentrifuge technique, into fractions of varying density. The packed red cell column was divided into three fractions; a bottom fraction rich in deformed cells or irreversibly sickled cells (ISC), with a very high mean corpuscular hemoglobin concentration (MCHC); a middle fraction containing cells with the highest content of fetal hemoglobin; and a top fraction containing reticulocytes and discoid cells but free of deformed cells. Oxygen affinity was shifted to the right in all layers (mean P(50) (pH 7.13)+/-1SD: top 46.3+/-2.9 mm Hg: middle 49.8+/-4.9 mm Hg; bottom 61.0+/-5.8 mm Hg) compared with normal blood (top 32.1+/-0.7 mm Hg: bottom 30.1+/-0.5 mm Hg). 2.3-DPG was increased in the top fraction, but was low or normal in the bottom fraction (top 21.8+/-3.4 mumol/g Hb: middle 17.7+/-2.2 mumol/g Hb; bottom 13.8+/-3.1 mumol/g Hb; normal whole blood 14.3+/-1.2 mumol/g Hb). The level of 2,3-DPG in top fractions could not account for the degree of right shift of P(50), and in the middle and bottom fractions the even greater right shifts were associated with lower levels of 2,3-DPG. Top fraction cells depleted of 2,3-DPG had a higher, but still abnormally low, oxygen affinity. A strong relationship was found between oxygen affinity and MCHC. The fractions with the greatest right shift in P(50) had the highest MCHC (top 32.4+/-2.0; middle 36.2+/-3.1; bottom 44.6+/-3.2 g/100 ml, respectively) and the plot of P(50) vs. MCHC showed a positive correlation (r = 0.90, P < 0.001). The red cell population in sickle cell anemia is not homogeneous but contains cells of widely varying Hb F content, 2,3-DPG, and hemoglobin concentration. Paradoxically, the cells with the lowest O(2) affinity have the lowest 2,3-DPG, but they also have the highest concentration of Hb S. The dense, deformed cell called the ISC is but the end stage in a process of membrane loss and consequent increase in hemoglobin concentration. The P(50) of Hb SS blood is, to a large extent, determined by the presence of these cells (r = 0.85, P < 0.001). Increased concentration of Hb S in the cell favors deoxygenation and crystallization even at relatively high P(o2). Lowered affinity for oxygen appears to be closely associated with Hb S concentration and not with 2,3-DPG content.

Relationship between the oxygen affinity and invitro sickling propensity of carbamylated sickle erythrocytes

Summary Incubation of Hb SS blood in vitro with NaCNO produces an increase in red cell oxygen affinity and a decrease in the propensity of the cells to sickle. The increase in oxygen affinity is dependent upon the extent of hemoglobin carbamylation. Carbamylated (2 moles of 14C cyanate incorporated per mole of hemoglobin) and control Hb SS cells sickle in vitro to an equal extent when the hemoglobin is deoxygenated to the same degree. Inhibition of the in vitro sickling phenomenon by hemoglobin carbamylation is in major part a result of the increased affinity of hemoglobin for oxygen.