Hereditary spherocytosis is associated with decreased pyruvate kinase activity due to impaired structural integrity of the red blood cell membrane.

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or ß-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2  = 0·15; slope = 9·09) and, less significantly, in HE (R2  = 0·021; slope = 8·92) when compared with other haemolytic disorders (R2  ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2  = 0·48; slope = -9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.

Unique profile for erythrocyte membrane acetylcholinesterase in hereditary spherocytosis.

Acetylcholinesterase of human erythrocytes from healthy donors and from patients with hematological disorders was analysed in a search for differential membrane parameters. Two substrates were used to estimate the exposure of acetylcholinesterase active site in the membrane: phenylacetate, a hydrophobic substrate, to determine total enzyme activity, and acetylcholine, an ionic substrate, to measure the externally reactive enzyme. The sensitivity of acetylcholinesterase to added stearic acid was also analysed. Three categories of the disorders studied were discerned: (a) The erythrocyte acetylcholinesterase profile was indistinguishable from normal control in beta-thalassemia minor and groups of patients with autoimmune hemolytic anemia or congenital dyserythropoietic anemia type II. (b) A marked decline in acetylcholinesterase with both substrates and reduced sensitivity to stearic acid were exhibited by the erythrocytes of paroxysmal nocturnal hemoglobinuria, beta-thalassemia major and other autoimmune hemolytic anemia and congenital dyserythropoietic anemia type II patients. Normal erythrocytes, either aged or pretreated to 50 degrees C, also showed similar characteristics. (c) Hereditary spherocytosis was singly differentiated by an elevated acetylcholinesterase activity with acetylthiocholine and by a vastly diminished sensitivity to stearic acid, while activity with phenylacetate was equal to control. This distinct profile may reflect the unique organization of the erythrocyte membrane in hereditary spherocytosis.

Phosphorylase activity in chronic erythremic myelosis.

Phosphorylase activity was detected in the cytoplasm of erythroid precursors of 6 of 7 patients with chronic erythremic myelosis (Di Guglielmo syndrome), in proerythroblasts and megaloblasts from 3 patients with pernicious anemia and in 2 patients with severe folate deficiency in neoplastic lymphocytes from 2 patients with acute lymphoblastic leukemia, and in 1 patient with leukemic lymphosarcoma. In all of these patients, most of the erythroid precursors and/or neoplastic lymphocytes contained increased amounts of glycogen when stained with the PAS reagent. Phosphorylase activity was not detected in erythroid precursors obtained from 6 presumed normal individuals or from 3 of 7 patients with a variety of other types of anemia in which the erythroid precursors were PAS-negative. Similarly, phosphorylase activity was absent in lymphocytes obtained from presumed normal individuals. Although the mechanisms responsible for the pathogenesis of PAS positivity are unclear, it is possible that the increased phosphorylase activity found in cells that are PAS-positive may reflect a disorder in the biosynthetic pathway of glycogen.

Partial characterization of cytosol and membrane-bound protein kinases in hereditary spherocytosis erythrocytes.

The protein kinase activity located in the cytosol of hereditary spherocytosis erythrocytes is due to multiple forms which can be resolved by Sepharose 6B filtration at high ionic strength into two fractions phosphorylating the whole casein on different sites. The membrane-bound protein kinases, solubilized by 0.7 M NaCl, display an elution volume from Sepharose column and a phosphorylation behaviour towards casein quite similar to those of the more retarded fraction of hemolysate. When compared with the multiple protein kinase forms from normal human erythrocytes, no significant difference has been found.

Red cell membrane phosphatidylinositol kinase activity in hemolytic anemias and myeloproliferative diseases.

Phosphatidylinositol kinase activity was determined in red cell membranes from 85 healthy individuals, 20 patients with hereditary hemolytic anemia and 24 patients with myeloproliferative disorder. Increased activity was found in all ten cases of sickle red disease and seven among ten cases of other hereditary hemolytic anemias. These increases had no correlation with the reticulocyte count nor with the red cell shape. An unexpected decreased activity was found in several cases of myeloproliferative disorders, especially in polycythemia vera, with a negative correlation with the reticulocyte count. The mechanism(s) and significance of the phosphatidylinositol kinase abnormalities in these different groups of diseases remain to determine.

Intra-erythrocytary enzymes before and after splenectomy.

The activity of the intraerythrocytary enzymes glucose-6-phosphate dehydrogenase, pyruvate kinase, glutathione reductase and ATPase was measured before and after splenectomy in 13 patients with congenital hemolytic anemia and 3 patients suffering from chronic thrombocytopenia. All patients were treated successfully, as reflected by clinical and basal hematological parameters. Glucose-6-phosphate dehydrogenase and pyruvate kinase were significantly depressed after splenectomy. It was not possible to set up prognostic criteria of splenectomy from the intraerythrocytary enzymes.

Study of a case with severe red-cell pyrimidine 5'-nucleotidase deficiency.

A deficiency of erythrocyte pyrimidine 5'-nucleotidase was found in a 23-year-old male suffering from severe congenital hemolytic disease. Results from exhaustive metabolic exploration are given and compared with reticulocyte-rich blood from subjects with auto-immune hemolytic disease. Evidence is given that the 20% apparent P5N residual activity corresponds to a non-specific acid phosphatase.

[Acetylcholinesterase of erythrocytes in various blood diseases]. / Atsetilkholinesteraza eritrotsitov pri nekotorykh zabolevaniiakh sistemy krovi

Appearance of low molecular components of acetylcholinesterase from erythrocyte membranes was found by gel filtration on Sephadex G-200 and polyacrylamide gel disc electrophoresis. Content of these components was distinctly increased within ghe acute period of Marchiafava-Micheli disease.

Membrane protein phosphorylation and protein kinases in normal and hereditary spherocytosis red cells.

Membrane protein phosphorylation by protein kinases in normal red cells takes place mainly on band 2 at the basal activity, and on bands 1 and 2.1 in the presence of cyclic 3':5'-adenosine monophosphate. Calcium precipitates preferentially bands 1 and 2.1 on extracted membrane proteins, and inhibit the membrane protein phosphorylation. Phosphorylation of endogeneous membrane proteins is diminished in red cells of some patients with hereditary spherocytosis (HS), partly corresponding reciprocally to MCHC, % spherocytes and reticulocytosis in peripheral blood of these patients, although the enzymatic activities of glyceraldehyde-3-phosphate dehydrogenase as a marker of the inner surface of red cell membranes are maintained normally in these red cells. The pattern of membrane protein fractions in HS red cells as endogeneous substrates for phosphorylation reactions is almost identical to that in normal red cells. Activities to phosphorylate casein or histone as exogeneous substrates are normal in HS red cell ghosts.

Calcium transport and adenosine triphosphatase activities of erythrocyte membranes in congenital spherocytosis.

Calcium transport from red cells was measured in seventeen patients with congenital or hereditary spherocytosis (HS). The efflux remained at a lower level in resealed ghost cells of patients than in normal cells both in the presence and absence of adenosine triphosphate (ATP). We studied the activities of Ca2+,Mg2+-ATPase, ouabain-sensitive Na+,K+-ATPase, Mg2+-ATPase and Ca2+-(spectrin-)ATPase in cell membranes prepared by washing the cells with hypotonic medium. The mean +/-SD Ca2+,Mg2+-ATPase/Mg2+-ATPase of HS patients was 3.34 +/- 1.06, and 2.81 +/- 0.42 in control subjects. Na+,K+-ATPase/Mg2+-ATPase was 2.38 +/- 0.38 in HS cells compared to 2.01 +/- 0.41 in normal cells. Ca2+-ATPase/Mg2+-ATPase of HS membranes was 0.57 +/- 0.18 and the control value 0.43 +/- 0.08. These data indicate calcium retention in the erythrocytes of HS patients in spite of increases in Ca2+,Mg2+-ATPase activity in the majority of patients.

Decreased red cell enolase activity in a 40-year-old woman with compensated haemolysis.

A 40-year-old woman splenectomized 17 years previously for hereditary haemolytic anaemia was investigated in our laboratory because of persistent conjunctival subicterus associated with compensated haemolysis. The results of the autohaemolysis and osmotic fragility tests were similar to those usually observed in hereditary spherocytosis. Red cell enzyme assays indicated a decreased amount of kinetically normal enolase. The genetic transmission of this defect could not be established since the only other affected member of the family was the proposita's father who died several years ago after splenectomy for an undefined haemolytic disorder.

Characteristics of peripheral blood monocytes in hereditary xerocytosis and spherocytosis.

Peripheral blood monocytes isolated from patients with congenital hemolytic anemia, hereditary xerocytosis and spherocytosis, demonstrated in vivo engulfment of red cell and platelet fragments. In addition, morphometric studies performed on these monocytes showed an increase in cytoplasmic/nuclear ratio as well as lysosome and phagosome volumes. The production of carbon dioxide from glucose-1-14C in abnormal monocytes was increased (15-80%) but the intracellular values of beta-glucuronidase and esterase activity were similar to control monocytes. Monocyte locomotion assessed in the presence of chemotactic stimuli was found significantly increased (73 +/- 12 monocytes/oil immersion fields vs. 46 +/- 5 for control monocytes). We concluded that the monocytes in hemolytic anemias associated with increased in vitro red cell fragmentation have some features resembling the 'stimulated' monocytes and that this alteration may be due to red blood cell fragment ingestion.

Study of a kindred with hereditary spherocytosis and glyceraldehyde-3-phosphate dehydrogenase deficiency.

A patient with hereditary spherocytosis (HS) was found to have glyceraldehyde-3-phosphate dehydrogenase (G3PD) deficiency by electrophoresis of the isolated red cell membranes on polyacrylamide gels with sodium dodecyl sulfate (PAGE SDS) as demonstrated by a diminished band 6 (G3PD) and confirmed by specific enzyme assay. Thirteen members of his family were studied: four were normal, two had HS alone, three had G3PD deficiency alone, and four had both HS and G3PD deficiency. G3PD deficient kindred members were probably heterozygous, since their red cell enzyme, while qualitatively normal, was present in half normal amounts. The G3PD deficiency alone was asymptomatic, and there was no evidence that the combination of HS with G3PD deficiency increased the clinical severity of the disease. However, G3PD deficiency, when combined with HS, was associated with an increase in protein band 4.5 on PAGE SDS. This band was also increased by incubation of normal red cells without glucose, and appeared to be a protein absorbed to the membrane as a consequence of metabolic stress. Hence, red cells with the combined abnormalities of both HS and G3PD deficiency showed signs of the exceptional metabolic stress to which they were exposed.

[Congenital hemolytic anemias]. / Angeborene hämolytische Anämien.

During the last years our knowledge of the structural composition of erythrocyte membranes has markedly increased. It is the aim of this review to discuss hereditary changes of the erythrocyte membrane of pathophysiological significance on the basis of these recent biochemical findings. The report particularly deals with structural proteins, such as spectrin, and its possible significance for corpuscular deformation (e. g. spherocytosis). In addition, the importance of hemoglobinopathies and hereditary enzymatic defects for the development of hemolysis is discussed.

Absence of one component of spectrin adenosine triphosphatase in hereditary spherocytosis.

The stimulation by calcium and magnesium of ATPase activity of isolated ghosts, of water-soluble protein (spectrin), and of residual vesicles, derived from normal erythrocytes and from hereditary spherocytes (H.S.), has been measured. The ATPase activity found in normal water-soluble protein (WSP) at low levels of calcium (0.1-2.0 mM) is essentially absent in H.S. water-soluble protein, but the ATPase activity with magnesium and with high levels of calcium (60-100 mM) is the same in H.S. and normal WSP. Compared to normal, H.S. ghosts have increased Mg2+-stimulated activity. This increased activity is retained by the sedimentable vesicles ("residue") after extraction of the ghosts with 0.025 mM EDTA. The Ca2+, Mg2+-ATPase associated with the calcium pump is not significantly different in H.S.

Human red cell membrane adenylate cyclase in normal subjects and patients with hereditary spherocytosis, sickle cell disease and unidentified hemolytic anemias.

We have investigated adenylate cyclase in ghosts from normal and pathologic human red blood cells. Basic parameters such as specific activity, apparent Michaelis constant (KMapp), and response to effectors: sodium fluoride (NaF), 5'-guanylyl imidodiphosphate (Gpp (NH)p), isoproterenol, and PGE1 were investigated. Basal and NaF-stimulated activities were measured in ghosts from patients with hereditary spherocytosis, sickle cell disease, and various unidentified hemolytic anemias. Both activities were increased in any of these pathologic conditions as compared with those of normal red blood cells. Normal values were found in patients with hereditary spherocytosis after splenectomy and in patients with heterozygous sickle cell disease. There was a good correlation between the reticulocyte count and adenylate cyclase activity in hereditary spherocytosis and in sickle cell disease with reticulocyte count lower than 10%. The enzyme activity of the first group was about three times that of the second group. There was no correlation at all in sickle cell disease with higher reticulocytosis and in the group of unidentified hemolytic anemias. These results suggest that increased adenylate cyclase activities are not specific of any of these diseases. In the patients with hereditary spherocytosis, the adenylate cyclase activity seems to be essentially related to younger mean age of red blood cell population while in the patients with sickle cell disease and in others with unidentified hemolytic anemias some additional factors might interfere directly with the enzyme and alter its activity.