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Biomedical subjects

R M Bookchin

Publications and source records attributed to R M Bookchin.

18 recordsLinked to original sources
PubMed1991

Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia.

To explore our hypothesis of a direct reticulocyte origin of irreversibly sickled cells (ISCs), we fractionated light, reticulocyte-rich, and discocyte-rich sickle anemia red cells on Stractan gradients, and examined the effects of deoxygenation-induced sickling, external Ca2+, acidification, and replacing external Na+ by impermeant N-methyl-D-glucamine (NMG+). Sickling permeabilized light reticulocyte-rich cells to cations (Na+, K+, and Ca2+) more than discocytes; without external Ca2+, Na+ influx matched K+ efflux, with stable cell volume; with Ca2+, many light, low hemoglobin (Hb) F reticulocytes dehydrated rapidly (preventable by quinine, a Ca2(+)-dependent K+ channel inhibitor). Acidification of oxygenated discocytes (high mean Hb F) and reticulocyte-rich fractions yielded denser, reticulocyte-enriched cells with lower Hb F (as in light reticulocyte or dense ISC-rich fractions). Light cells shrank when NMG+ replaced Na+, supporting predictions of a Na(+)-dependent volume control system. Demonstration of sickling-induced, Ca2(+)-dependent dehydration of Hb F-free reticulocytes, and conservation of acid-stimulated K:Cl cotransport among low Hb F, reticulocyte-enriched cells in discocyte fractions support the hypothesis. Ancillary new findings included heparin stimulation of sickling-induced Na+ and K+ permeabilizations, and Ca2+ inhibition of the Na+ leak.

Anemia, Sickle Cell↗
PubMed1990

Effect of osmotic lysis and resealing on red cell structure and function.

We have recently modified the dialysis tubing osmotic lysis and resealing method to examine the role of intracellular red blood cell (RBC) antioxidants. However, the potential effect of resealing on the RBC was not fully investigated. This study examined a number of cellular characteristics to determine the effects of physical lysis and resealing on the RBC. Following resealing, RBC exhibited normal morphology and at most only slight alterations in mean cell volume and mean cell hemoglobin concentration. RBC density distribution was significantly affected by resealing with increased populations of both light and dense cells, though the mean cell density was similar to that of control cells. Endogenous enzyme activities and adenosine triphosphate (ATP) concentration were unaffected by the resealing procedure. While reduced glutathione (GSH) concentration was decreased by 15%, RBC oxidant sensitivity was found to be unaltered. Cellular deformability of the resealed RBC was 80% to 90% that of the control cells. Membrane phospholipid and fatty acyl composition of the resealed RBC were unaffected when compared with matched control samples. Membrane transport, permeability, and Ca2(+)-mediated cellular vesiculation were minimally altered by resealing. Finally, entrapment of fluorescent compounds demonstrated that greater than 95% of the resealed RBC had incorporated exogenous agents. In summary, the osmotic lysis and resealing method described resulted in only minor changes in cellular characteristics while allowing for the efficient loading of compounds to which the RBC membrane is normally impermeable. Consequently, this method provides great potential for the selective modification of erythrocyte constituents in order to further define their roles within the RBC.

Adenosine Triphosphate↗
PubMed1979

Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S.

We have previously found that the inhibitory effect of hemoglobin F (Hb F) on the polymerization of Hb S proceeds via the formation of asymmetrical hybrid tetramers of the type alpha2betasgamma. Examination of the gelling properties of binary mixtures of Hb S and several Hb variants now shows that, among the gamma chain amino acid residues that differ from those of the beta chain, residues gamma80 (EF4) and gamma87 (F3) are at least partly responsible for this inhibition. Furthermore, we find that mixing Hb A2(alpha2delta2) with Hb S strongly inhibits gelling to an extent similar to that seen with Hb S/Hb F mixtures; this inhibition is attributable to amino acid differences between the delta and beta chain sequences at positions delta22 (B4) and delta87 (F3). Therefore, residues 22, 80, and 87 of the beta chain appear to be involved in intermolecular contact sites that stabilize the deoxy Hb S polymers.

Amino Acid Sequence↗
PubMed1979

An increased Bohr effect in sickle cell anemia.

Recent findings that hemoglobin S gelation and sickling are pH-dependent and also influence oxygen affinity suggested that the red cells containing this hemoglobin variant might show an abnormal Bohr effect. We therefore studied the effects of pH variation on the in vitro oxygen affinity of whole blood from persons with sickle cell anemia (SS) and normal donors (at 37 degrees C and constant carbon dioxide tension of 40 mm Hg). The Bohr effect in SS blood was greatly increased only between blood pH 7.4 and 7.2 (cell pH 7.2 and 7.0, a shift that strongly affects gelation), with delta log p50/deltapH= - 0.92 to -0.99 (normal = -0.42 to -0.46). Thus a drop in SS blood pH below 7.4 in tissue capillaries yields twice the normal decrease in oxygen affinity and a large release of oxygen from red cells, whose risk of sickling is high. Even mild transient acidosis would seem hazardous for patients with sickling disorders.

Anemia, Sickle Cell↗
PubMed1979

Hemoglobin Crete (beta 129 ala leads to pro): a new high-affinity variant interacting with beta o -and delta beta o -thalassemia.

Hemoglobin Crete, beta129 (h7)ala leads to pro, is a new mutant hemoglobin (Hb) with high oxygen affinity that was discovered in a Greek family in various combinations with beta- and deltabeta-thalassemia. The propositus, who presented an unusual clinical picture of an "overcompensated" hemolytic state, with erythrocytosis, splenomegaly, abnormal red cell morphology, and marked erythroid hyperplasia, appeared doubly heterozygous for Hb Crete and deltabeta-thalassemia. His red cells contained 67% Hb Crete and 30% Hb F, and the combination of these two hemoglobins resulted in a blood P50O2 of 11.2 mm Hg. A brother with Hb Crete trait (38% Hb Crete, 56% Hb A, blood P50O2 23.0 mm Hg) did not have significant erythrocytosis. Purified Hb Crete was heat-unstable and exhibited a high oxygen affinity, and a normal Bohr effect. We postulate that the beta 129 proline substitution disrupts the H helix, perturbing nearby residues involved in alpha 1 beta 1 contact sites of the Hb tetramer.

Adult↗
PubMed1979

Deoxyhemoglobin S gelation and insolubility at high ionic strength are distinct phenomena.

Deoxyhemoglobin S in solution forms a solid phase either in solutions of high ionic strength or in solutions of low ionic strength but a high hemoglobin concentrations. Presently, both techniques are in use to study various hemoglobin interactions which might occur during sickling of Hb S-containing red cells. When Hb S is treated with either nitrogen mustard or cyanate, there is no correlation between high-ionic-strength salting out and low-ionic-strength gelation studies. These results suggest that salting out studies are not equivalent to the assays that depend on the gelation of polymerization of Hb S.

Alkylation↗
PubMed1978

Kinetics of HB S gelation. Effect of alkylureas, ionic strength and other hemoglobins.

Using a light scattering (turbidity) method to estimate the delay time of gelation of deoxy hemoglobin S hemolysates, we have examined the effects of various alkylureas, variations in ionic strength by addition of NaCl, and admixture with other hemoglobins on gelation kinetics. Each of these factors substantially prolonged the delay times to different extents, but the dependence of the delay times on a high power of the hemoglobin concentration varied only slightly. These findings suggest that the events preceding gelation, most likely the formation of nuclei, are affected by these factors, but the critical nuclear size for gelling is fairly constant. Parallel changes indicated good qualitative correlation between gelation, delay times, minimum gelling concentrations of solutions of mixed hemoglobins and kinetics of sickling of red cells containing these mixtures, with the exception of hemoglobin Charlem trait cells the sickling kinetics of which were slower than predicted by the solution properties.

Gels↗
PubMed1977

Ligand kinetics of hemoglobin S containing erythrocytes.

Oxygen uptake of fully deoxygenated sickle (SS) erythrocytes is slower than that of normal (AA) erythrocytes, as demonstrated by the half-times of the overall oxygenation reactions: at 25 degrees in an isotonic phosphate buffer the normal red cells have a t1/2 = 82 +/- 4.7 msec, as compared to sickle red cells where t1/2 = 135 +/- 17.6 msec. The effects of temperature, extracellular osmolality, and the presence of an antisickling agent (n-butylurea) on the rate of red cell oxygenation strongly suggest that the differences in oxygenation rates encountered with sickle red cells is directly related to the intracellular polymerization of deoxyhemoglobin S.

Antisickling Agents↗
PubMed1976

Hemoglobin Beth Israel. A mutant causing clinically apparent cyanosis.

We found that an abnormal hemoglobin with a very low oxygen affinity was responsible for overt cyanosis in an otherwise healthy adolescent. Hemoglobin Beth Israel, in which serine replaces the asparagine residue normally present at position 102 (G4) of the beta-polypeptide chain, was associated with normal blood counts and no apparent exercise intolerance in the heterozygous carrier. Cyanosis resulted from a drastically right-shifted oxygen dissociation curve, whose position and shape could account for the absence of "physiologic" anemia. The whole-blood oxygen tension at 50 per cent oxygen saturation was 88 mm Hg (normally 26 +/- 1 mm Hg), and the arterial blood was only 63 per cent saturated with oxygen despite a normal oxygen tension of 97 mm Hg. The hemolysate showed a low oxygen affinity but normal Bohr effect. Unexplained cyanosis, particularly in association with normal arterial oxygen tension should prompt a search for an abnormal hemoglobin, which may obviate the need for invasive diagnostic procedures.

Adolescent↗
PubMed1976

Determinants of red cell sickling. Effects of varying pH and of increasing intracellular hemoglobin concentration by osmotic shrinkage.

The effects of varying pH and of increasing intracellular hemoglobin (Hb) concentration on red cell sickling and oxygen affinity were studied in whole blood from persons with sickle cell anemia (SS) and sickle cell trait (SA). Small increases in SS blood pH inhibited sickling, and small reductions in both SS and SA blood pH promoted sickling far more than accounted for by the Bohr effect. Sickling behavior correlated with minimum gelling concentrations (MGC) of deoxygenated hemolysates without 2,3-diphosphoglycerate. MGC values fell sharply when pH was lowered from 7.25 to 7.15 for HbS and from 7.15 to 6.90 for SA hemolysates, suggesting effects on specific ionic interactions involved in Hb gelation. Possible clinical counterparts are acute metabolic acidosis and alkalosis (prior to change in red cell 2,3-diphosphoglycerate), where the Bohr effect and oxygen affinity-independent effects of pH alterations on sickling would be additive. Osmotic shrinkage of HbS-containing red cells produced a large fall in oxygen affinity and a marked increase in sickling independent of that fall. The oxygen affinity and sickling properties of SA cells whose MCHC was raised to 40 per cent resembled those of unaltered SS cells, supporting a relationship between molecular aggregation of Hb and low oxygen affinity. Sickling of aerated SS cells in hypertonic saline depended upon partial Hb desaturation due to lowered oxygen affinity. Thus osmotic shrinkage of HbS-containing cells acts synergistically with partial deoxygenation to promote sickling. These conditions are present in the renal medulla, but may occur elsewhere in severe hyperosmolar states.

Adult↗
PubMed1976

The conformational requirements for the mechanical precipitation of hemoglobin S and other mutants.

The mechanical stability of human hemoglobin mutants was studied for the specific effects of single and double amino acid substitutions, the ligand state of each chain, and the effect of hybrids between oxy and cyanmet partners on precipitability. It was found that the beta6Glu leads to Val and the beta73 Asp leads to Asn mutations increased the degree of mechanical precipitation in the liganded but not in the deoxy form. When these mutations occurred on the same chain, the effects were approximately additive. Heat labile mutants such as Hb Gun Hill and Hb Leiden exhibited mechanical instability, but probably through a different mechanism, as very little dependence on ligand state was apparent. Studies with valency hybrids of HbS(alpha2 betas2-and-alpha2 betas2 where = cyanmet) revealed that instability was primarily determined by the state of the betas chain, which must be liganded to confer instability on the tetramer. A good agreement between surface activity and mechanical precipitability of these mutants has been found.

Chemical Precipitation↗
PubMed1976

Molecular and cellular effects of antisickling concentrations of alkylureas.

Alkylureas are capable of inhibiting sickling in vitro and the gelation of solutions of hemoglobin S at concentrations between 0.05 and 0.1 M with increasing effectiveness that is directly proportional to the length of the alkyl chain (butyl greater than propyl greater than ethyl greater than methyl). 6The inhibitory effect is independent of pH between 6.5 and 7.5 and is a process driven by entropy. The alkylureas at concentrations of 0.1 M have minimal effects on several erythrocyte functions. Oxygen equilibria, osmotic fragility, reduced glutathione content, and glutathione reductase activity are totally unaffected, while pyruvic kinase activity is decreased only by butylurea by about 20%, and glucose-6-phosphate dehydrogenase activity is decreased progressively to a maximum of 30% in direct proportion to the length of the alkyl chain. Alkylureas not only inhibit sickling but are also capable of desickling erythrocytes that have been maintained in the deoxygenated state. They have little effect on several erythrocyte functions at antisickling concentrations, but their toxicity must be evaluated before they can be examined as potential therapeutic agents for the treatment or prevention of acute episodes in sickle cell anemia.

Erythrocytes, Abnormal↗
PubMed1975

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.

Anemia, Sickle Cell↗