Direct intracellular measurement of deoxygenated hemoglobin S solubility

Blood ◽  
2001 ◽  
Vol 98 (3) ◽  
pp. 883-884 ◽  
Author(s):  
Mary E. Fabry ◽  
Laurephile Desrosiers ◽  
Sandra M. Suzuka
Keyword(s):  
Transgenic Mouse ◽  
Hemoglobin Concentration ◽  
Red Cells ◽  
Red Cell ◽  
Hemoglobin S ◽  
Physiological Conditions ◽  
Polymer Formation ◽  
Solution Studies ◽  
Deoxygenated Hemoglobin

Abstract The solubility of deoxygenated hemoglobin S (HbS), which is the concentration of fully deoxygenated HbS in equilibrium with polymer (CSAT), is a factor that determines in vivo polymer formation. However, measurement of CSAT is usually performed under conditions that are far from physiological. In solution studies of HbS by Benesch et al, it was demonstrated that p50, the point at which hemoglobin is half-saturated with oxygen, is proportional to the amount of polymer formed and that it may be used to measure CSAT. This method has been extended to measure CSAT in intact red cells by varying extracellular osmolarity, which, in turn, varies intracellular hemoglobin concentration. This method measures intracellular CSAT under physiological conditions with intact red cell contents and can be applied to human and transgenic mouse red cells. The principle is demonstrated by measuring p50 as a function of extracellular osmolarity for AA, SS, and AS red cells.

LACK OF EFFECT OF CORTICOSTEROIDS ON THE IN VIVO DISAPPEARANCE OF SULFHYDRYL-INHIBITED AND ANTIBODY-COATED ERYTHROCYTES

1964 ◽  
Vol 47 (3_Suppl) ◽  
pp. S28-S36
Author(s):  
Kailash N. Agarwal
Keyword(s):  
Red Cells ◽  
List Type ◽  
Red Cell

ABSTRACT Red cells were incubated in vitro with sulfhydryl inhibitors and Rhantibody with and without prior incubation with prednisolone-hemisuccinate. These erythrocytes were labelled with Cr51 and P32 and their disappearance in vivo after autotransfusion was measured. Prior incubation with prednisolone-hemisuccinate had no effect on the rate of red cell disappearance. The disappearance of the cells was shown to take place without appreciable intravascular destruction.

pH environmental of red cells in the spleen

1976 ◽  
Vol 231 (6) ◽  
pp. 1672-1678 ◽  
Author(s):  
MJ Levesque ◽  
AC Groom
Keyword(s):  
Splenic Tissue ◽  
Red Cells ◽  
Red Cell ◽  
Cat Spleen ◽  
Flow Through ◽  
Red Pulp ◽  
Reduced Flow

Intrasplenic pH in vivo was deduced from measurements on blood drained from cat spleen during contraction with the inflow occluded. The pH of blood in the red pulp is normally 7.20, but stasis or reduced flow through the pulp causes pH to fall toward 6.8. The splenic pulp contains blood of high hematocrit. To evaluate the role of buffering by the red cells themselves, intrasplenic p/ in red cell-free spleens was, therefore, estimated atering and leaving the spleen during red cell washout. At inflow pH less than 6.8 the outflow pH was raised, at inflow pH = 6.8 there was no change, b,t at inflow pH greater than 6.8 the outflow pH was lowered. These results indicate that the pH environment of red cells in the spleen results indicate that the pH environment of red cells in the spleen results from the interplay of two separate factors: i) pH-determining elements of the splenic tissue that buffer at 6.8, and ii) buffering provided by red cells passing through the pulp.

Red cell function at extreme altitude on Mount Everest

1984 ◽  
Vol 56 (1) ◽  
pp. 109-116 ◽  
Author(s):  
R. M. Winslow ◽  
M. Samaja ◽  
J. B. West
Keyword(s):  
Cell Function ◽  
Blood Gases ◽  
Hemoglobin Concentration ◽  
Respiratory Alkalosis ◽  
Blood Collection ◽  
Red Cell ◽  
Arterial Blood ◽  
Extreme Altitude ◽  
2,3 Dpg

As part of the American Medical Research Expedition to Everest in 1981, we measured hemoglobin concentration, red cell 2,3-diphosphoglycerate (2,3-DPG), Po2 at which hemoglobin is 50% saturated (P50), and acid-base status in expedition members at various altitudes. All measurements were made in expedition laboratories and, with the exception of samples from the South Col of Mt. Everest (8,050 m), within 2 h of blood collection. In vivo conditions were estimated from direct measurements of arterial blood gases and pH or inferred from base excess and alveolar PCO2. As expected, increased 2,3-DPG was associated with slightly increased P50, when expressed at pH 7.4. Because of respiratory alkalosis, however, the subjects' in vivo P50 at 6,300 m (27.6 Torr) was slightly less than at sea level (28.1 Torr). The estimated in vivo P50 was progressively lower at 8,050 m (24.9 Torr) and on the summit at 8,848 m (19.4 Torr in one subject). Our data suggest that, at extreme altitude, the blood O2 equilibrium curve shifts progressively leftward because of respiratory alkalosis. This left shift protects arterial O2 saturation at extreme altitude.

scholarly journals Effect of oxalate and malonate on red cell metabolism

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1389-1393
Author(s):  
E Beutler ◽  
L Forman ◽  
C West
Keyword(s):  
Pyruvate Kinase ◽  
Cell Metabolism ◽  
Inhibitory Effect ◽  
Red Cells ◽  
Red Cell ◽  
Acid Analogue ◽  
2,3 Dpg

The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

scholarly journals The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1427-1431 ◽  
Author(s):  
N Fortier ◽  
LM Snyder ◽  
F Garver ◽  
C Kiefer ◽  
J McKenney ◽  
...  
Keyword(s):  
Protein Complex ◽  
Sodium Dodecyl ◽  
Red Cells ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Cellular Dehydration ◽  
Thalassemia Minor ◽  
Membrane Rigidity

Abstract In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

scholarly journals The Role of Red Cell Energy Metabolism in the Generation of Irreversibly Sickled Cells In Vitro

Blood ◽  
1973 ◽  
Vol 42 (6) ◽  
pp. 835-842 ◽  
Author(s):  
Michael Jensen ◽  
Stephen B. Shohet ◽  
David G. Nathan
Keyword(s):  
Energy Metabolism ◽  
Calcium Metabolism ◽  
Red Cells ◽  
Red Cell ◽  
Hemoglobin S ◽  
Cell Energy ◽  
Incubation Buffer ◽  
Membrane Defect

Abstract An acquired membrane defect is believed to be responsible for the maintenance of the sickled shape in oxygenated irreversibly sickled cells (ISC), because the hemoglobin S in these cells is not in the aggregated, "sickled" state. In the present study, it is demonstrated that the acquisition of the membrane defect in vitro depends on cellular metabolism. Only if cellular ATP is almost completely depleted while the cells are sickled, do they become unable to resume the biconcave disk shape upon reoxygenation. If calcium is omitted from the incubation buffer, ISCs are not generated despite metabolic depletion. This suggests an action of ATP mediated through calcium metabolism similar to that which prevents membrane stiffening in normal red cells. No ISCs were produced by repeated sickling and unsickling. Thus, a membrane alteration occurring as a consequence of metabolic depletion seems to be a more important factor in the generation of ISC than sickling-unsickling induced fragmentation.

scholarly journals Reticulocyte Survival in Sickle Cell Anemia: Effect of Cyanate

Blood ◽  
1972 ◽  
Vol 40 (5) ◽  
pp. 733-739 ◽  
Author(s):  
Blanche P. Alter ◽  
Yuet Wai Kan ◽  
David G. Nathan
Keyword(s):  
Cell Survival ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Red Cells ◽  
Red Cell ◽  
Hemoglobin Molecule ◽  
Red Cell Survival ◽  
And Control

Abstract Cyanate prevents sickling in vitro and apparently prolongs the survival of 51Cr-tagged sickle erythrocytes in vivo. Cautious interpretation is required because the effects of cyanate on 51Cr binding to sickle and fetal hemoglobin-containing red cells are unknown, and comparison of the effect of cyanate on sickle red cell survival to control red cell survival must be performed sequentially. We have studied the survival of sickle reticulocytes utilizing radioactive amino acids that are incorporated into hemoglobin. Two informed adult patients with sickle cell disease were studied. In each study, two 50-ml samples of blood were incubated separately with 14C- and 3H-leucine for 2 hr, after which 50 mM cyanate was added to one aliquot for 1 hr. The cells were then washed and reinfused. Frequent venous samples were obtained, and the specific activities of 14C and 3H in the hemoglobin were followed. The t ½ of the carbamylated cells was tripled, but remained below normal. This method provides a generally useful measurement of the influence of drugs bound to red cells on reticulocyte lifespan. The labels are incorporated into the hemoglobin molecule of the reticulocyte, and simultaneous comparison of the survivals of the same cohort of drug-treated and control cells is achieved.

scholarly journals Effect of oxalate and malonate on red cell metabolism

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1389-1393 ◽  
Author(s):  
E Beutler ◽  
L Forman ◽  
C West
Keyword(s):  
Pyruvate Kinase ◽  
Cell Metabolism ◽  
Inhibitory Effect ◽  
Red Cells ◽  
Red Cell ◽  
Acid Analogue ◽  
2,3 Dpg

Abstract The addition of oxalate to blood stored in Citrate-phosphate-dextrose (CPD) produces a marked improvement in 2,3-diphosphoglycerate (2,3-DPG) preservation; an increase in 2,3-DPG levels can also be documented in short-term incubation studies. Oxalate is a potent in vitro inhibitor of red cell lactate dehydrogenase, monophosphoglycerate mutase, and pyruvate kinase (PK). In the presence of fructose 1,6-diphosphate the latter inhibitory effect is competitive with phospho(enol)pyruvate (PEP). Determination of the levels of intermediate compounds in red cells incubated with oxalate suggest the presence of inhibition at the PK step, indicating that this is the site of oxalate action. Apparent inhibition at the glyceraldehyde phosphate dehydrogenase step is apparently due to an increase in the NADH/NAD ratio. Oxalate had no effect on the in vivo viability of rabbit red cells stored in CPD preservatives for 21 days. Greater understanding of the toxicity of oxalate is required before it can be considered suitable as a component of preservative media, but appreciation of the mechanism by which it affects 2,3-DPG levels may be important in design of other blood additives. Malonate, the 3-carbon dicarboxylic acid analogue of oxalate late did not inhibit pyruvate kinase nor affect 2,3-DPG levels.

scholarly journals Fragility and structure of hemoglobin S fibers and gels and their consequences for gelation kinetics and rheology

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 573-579 ◽  
Author(s):  
RW Briehl ◽  
AE Guzman
Keyword(s):  
Delay Time ◽  
Hemoglobin Concentration ◽  
Differential Interference Contrast ◽  
Cell Disease ◽  
Mechanical Perturbation ◽  
Hemoglobin S ◽  
Gelation Kinetics ◽  
A Value ◽  
Cross Links

Abstract Pathogenesis in sickle cell disease depends on whether red blood cells can pass the microvasculature during the delay time before hemoglobin S gelation and cell rigidification occur. Here we observe individual hemoglobin S fibers by differential interference contrast (DIC) microscopy and show that hemoglobin S gels and fibers are fragile and easily broken by mechanical perturbation, and that breakage results in vast acceleration of gelation kinetics due to the creation of new, growing fiber-ends. Hence, in vivo this may be an important factor, in addition to hemoglobin concentration and degree of deoxygenation, that governs delay time and pathogenesis. Pathogenesis also depends on gel rheology and cell rigidification, which depend on fiber cross-linking. We show different mechanisms by which X-shaped, Y-shaped, and “zippering” cross-links form. Finally, we estimate the “on” rate constant for fiber growth to be about 200 mmol/(L.s) and obtain a value for the heterogeneous nucleation rate at 13.5 mmol/L heme.

scholarly journals Near-normal circulatory survival of rabbit red cells exposed to high levels of Ca and ionophore in vitro

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 220-223 ◽  
Author(s):  
RM Bookchin ◽  
EF Jr Roth ◽  
VL Lew
Keyword(s):  
Adenosine Triphosphate ◽  
Indirect Evidence ◽  
Red Cells ◽  
Ionophore A23187 ◽  
Red Cell ◽  
L Cells

Abstract The belief is widely held, on the basis of indirect evidence, that a substantial, even brief elevation of red cell Ca content must result in a marked shortening of circulatory survival. To test this notion directly, we exposed rabbit red cells in vitro to the ionophore A23187 and Ca so as to produce sustained uniform cell Ca levels of 40 to 360 mumol/L cells for one to 60 minutes, and compared the survival of the Ca-loaded cells in vivo with that of ionophore-treated controls, simultaneously, in the same rabbits. Despite marked reductions in cell adenosine triphosphate and dehydration of the Ca-exposed cells prior to reinfusion, the majority of cells, all of which had experienced these high cytoplasmic Ca levels, showed normal or near-normal survival in the circulation.

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