Hereditary nonspherocytic hemolytic anemia due to a new hexokinase variant with reduced stability

A 27-year-old woman with severe chronic hemolytic anemia was found to have reduced red cell hexokinase activity when the degree of reticulocytosis was considered. This enzyme had normal pH-dependent activity, normal Km for glucose, fructose, and mannose, normal Km for Mg adenosine triphosphate (ATP)2- and Ki for glucose-1,6-diphosphate. Furthermore, the pH-dependence and orthophosphate dependence of Ki for glucose-1,6-diphosphate were normal. However, this hexokinase was inactivated rapidly at 44 degrees C. No abnormalities were found in the red cell hexokinase isozymic pattern when it was compared with the profile obtained from cells of similar age. The hexokinase specific activity was reduced in all the red blood cell fractions obtained by density gradient ultracentrifugation; a marked difference in the distribution of cells through the gradient was evident. Among the glycolytic intermediates, a significant decrease of 2,3- diphosphoglycerate was evident. ATP and glucose 6-phosphate were also reduced when compared with cells of similar. Glucose consumption of the hexokinase-deficient cells decreased, but the rate of glucose metabolized through the hexose monophosphate shunt was unchanged. Although the total hexokinase activity in lymphocytes was only reduced by 37%, a marked hexokinase deficiency was detected in blood platelets (20% to 25% of normal activity). The parents and one of two siblings of the patient were heterozygous for the defect, with 66% to 74% of normal erythrocyte hexokinase activity and reduced heat stability of the enzyme. These results, when compared with those obtained in previously reported cases of hexokinase deficiency, provide further evidence of the broad phenotypic variability that characterizes this disorder. Furthermore, it is suggested that failure of energy generation is probably the primary cause of hemolytic anemia in hexokinase deficiency.     

Generalized hexokinase deficiency in the blood cells of a patient with nonspherocytic hemolytic anemia

In a patient with nonspherocytic hemolytic anemia, a hexokinase deficiency was detected in the red cells (residual activity about 25% of normal) and in blood platelets (20%-35% of normal activity). Although the total hexokinase activity in lymphocytes was normal, the amount of hexokinase type I was decreased to about 50% of normal. However, the deficiency was compensated for by the appearance of type III hexokinase. Compartmentation studies with controlled digitonin- induced cell lysis showed that this type III enzyme was localized in the cytosol, while almost all hexokinase activity in normal lymphocytes is particulate. No abnormal lymphocyte functions could be detected. The patient was homozygous for the defect. The parents and three of five sibs of the patient were apparently heterozygous with residual activities of 50%-67% of normal in their red cells, but did not show any clinical signs of hexokinase deficiency. The variant enzyme had a slightly decreased affinity for MgATP2- and a strongly increased inhibition constant for glucose-1,6-P2. Affinity for glucose, heat stability, and pH optimum were normal. In the electrophoretic pattern of red cell hexokinase, only one subtype of hexokinase I could be detected, while in normal red cells, at least three subtypes are present. In the heterozygous individuals, no enzymatic abnormalities could be detected, except for an aberration in the electropherogram of one sib.     

Human erythrocyte hexokinase deficiency: a new variant with abnormal kinetic properties

A 14-month-old child who had a haemolytic episode when he was 5 years old, and with psychomotor retardation, was found to have decreased red cell hexokinase activity. The mutant enzyme was characterized by an increased affinity for glucose associated with an increased inhibition constant for glucose-1,6-diphosphate. Affinity for Mg ATP2-, heat stability and pH-optimum were normal. The isozymic pattern of the red cell enzyme was normal but all the molecular forms were present in reduced amounts. The kinetics of decay of hexokinase during cell ageing was also normal. Glucose consumption of the hexokinase deficient cells was 60-65% of the controls while the amount metabolized through the hexose monophosphate shunt was unchanged. Red cell 2,3-diphosphoglycerate and glucose-6-phosphate levels were normal in the proband but reduced in the erythrocytes of his parents, who were heterozygous for the defect but had normal haematological data. Comparison with the 13 previously reported cases of hexokinase deficiency confirms the broad phenotypic variability that characterizes this disorder.     

Evidence for Abnormal Red Cell Population in Refractory Sideroblastic Anemia

Red cell enzymes were studied in 11 patients with refractory sideroblasticanemia. Eight of the cases were of the primary idiopathic type while threehad an underlying (?) disease. The red cells were separated into several densitygroups by different flotation with phthalate ester mixtures. The glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, hexokinase andpyruvate kinase activities and adenosine triphosphate contents were determined in each fraction, with special attention to the lightest and densestfractions.

It was found that the refractory anemia patients have a population of abnormal light red cells with a very high enzymatic activity, especially of hexokinase, a normal adenosine triphosphate content but relatively reduced surfacecharge. This abnormal population which so far seems typical to this disease,is probably another reflection of the disordered red cell proliferation andmaturation seen in refractory anemia.

Submitted on August 27, 1969 Accepted on December 8, 1969     

Glycolysis and Glycolytic Enzyme Activity of Aging Red Cells in Man

Hexokinase, aldolase, glyceraldehyde-₃-phosphate dehydrogenase, pyruvate kinase and glutamic-oxalacetic transaminase activities, and the rate of glycolysis have been measured in red cells from seven adult males responding to repeated phlebotomy or erythropheresis with iron replacement. The change in median age of the red cell populations of these men was estimated from the measured rates of red cell loss and erythropoietic replacement. These enzyme activities and glycolytic rates were measured also in a patient with aplastic anaemia in whom transfused red cells were aging at a known rate.
Hexokinase, aldolase and transaminase activities in the youngest red cell populations were four times greater than the corresponding levels in normal aged cell populations. Pyruvate kinase activity was two or three times higher than normal, but glyceraldehyde-₃-phosphate dehydrogenase activity was not significantly increased in the youngest cell populations. The rate of glycolysis in intact cells and of lactate formation from glucose or glucose-6-phosphate in coenzyme-supplemented haemolysate showed a two-fold increase above normal in the youngest cell populations. Subnormal enzyme activity in older cells was observed only for transaminase.
The deterioration of hexokinase and aldolase activity with red cell aging is sufficiently rapid and the activity of these enzymes is normally sufficiently low to indicate a severe glycolytic limitation in old red cells. Such limitation in glycolysis may impair red cell viability and be the determining factor in normal red cell life span.
Measurement of activity of hexokinase, aldolase, or glutamic-oxalacetic transaminase in an unknown red cell population should indicate the median age of the cell population much sooner than such information can be obtained by measurement of red cell survival.     

Two cases of red cell aldolase deficiency associated with hereditary hemolytic anemia in a japanese family

Two cases of red cell aldolase deficiency associated with congenital nonspherocytic hemolytic anemia are reported. The proband is a fourteen-month-old Japanese boy. Consanguineous marriage was not proven but probable in this family, as the parents were born in the same small island. The proband had moderate to mild anemia aggravated by upper respiratory infections, 1 cm hepatomegaly and 2.5 cm splenomegaly, but was unremarkable in other respects and has thus far not shown mental or growth retardation. He did not have dysmorphic features. The red cell aldolase activity was 6% of the normal mean. The enzyme was unstable with respect to heat, and Km for fructose 1,6-diphosphate (F-1,6-DP) was high. The parents and other heterozygotes showed intermediate activity between that of the proband and that of normal subjects. Red cell F-1,6-DP concentration in this case was remarkably increased. Red cell glucose consumption, and lactate formation, as well as hexose monophosphate shunt activity, were decreased as compared with a comparable reticulocyte-rich hereditary spherocytosis patient. Hexose monophosphate shunt activity was assumed to be decreased due to inhibition of glucose 6-phosphate dehydrogenase by a high concentration of F-1,6-DP in his red cells. As a result of family study, another homozygous aldolase deficiency case associated with hemolytic anemia was found. He is 13 years old and a nephew of the proband's paternal grandmother. His hemolytic anemia also is moderate to mild and aggravated by upper respiratory infections. He does not seem to have mental or growth retardation, nor does he possess dysmorphic features.     

A case of hemolytic anemia due to erythrocyte pyrimidine 5'-nucleotidase deficiency

A propositus, the offspring of a first-cousin marriage, was presented with severe hemolytic anemia, splenomegaly, jaundice, and growth retardation. Marked basophilic stippling of erythrocytes was shown by Wright's stain. Erythrocyte 5'-nucleotidase activity was found markedly decreased, whereas red blood cell glucose-6-phosphate dehydrogenase activity was elevated as the reduced glutathione level. His growth and anemia improved following splenectomy. His sister was also similarly affected.     

Red blood cell hexokinase in Fanconi's anemia

The activity of ten red blood cell enzymes, including hexokinase, has been measured in 6 Fanconi's anemia patients. In disagreement with previous reports, in no instance were reduced or increased hexokinase levels found. Furthermore, the hexokinase isozymic pattern, thermostability, pH dependence of activity and kinetic properties were also in the normal range.     

Elevated pyruvate kinase activity in patients with hemolytic anemia due to red cell pyruvate kinase "deficiency"

Two patients with non-spherocytic hemolytic anemia were found to have elevated red blood cell pyruvate kinase activities commensurate with the decreased mean red cell age, but the residual pyruvate kinase had marked kinetic abnormalities. Accumulation of metabolic intermediates before pyruvate kinase and reduced levels of activity of the red blood cells of the parents of both patients supported the diagnosis of an inherited abnormal pyruvate kinase causing hemolytic anemia. Although it was observed in two unrelated persons, review of enzyme assays performed on the red blood cells of 651 patients with hereditary non-spherocytic hemolytic anemia suggests that this occurrence is rare.     

Combined glucose-6-phosphate dehydrogenase and glucosephosphate isomerase deficiency can alter clinical outcome

Glucosephosphate isomerase (GPI) deficiency in humans is an autosomal recessive disorder, which results in nonspherocytic hemolytic anemia of variable clinical expression. A 4-year-old female with severe congenital hemolytic anemia had low red cell GPI activity of 15.5 IU/g Hb (50% of normal mean) indicating GPI deficiency. Subsequent DNA sequence analysis revealed a novel homozygous 921C to G mutation in the GPI gene sequence, predicting a Phe307 to Leu replacement. Strikingly, the red cell GPI activity in this patient was higher than that found in a second patient expressing the same GPI variant, with a more severe clinical phenotype. We propose that the hemolysis in the first patient may be modified by an accompanying deficiency of glucose-6-phosphate dehydrogenase (G6PD). The proband's red cell G6PD activity was reduced at 4.5 IU/g Hb (50% of normal mean) and molecular studies revealed heterozygosity for the G6PD Viangchan mutation and a skewed pattern of X-chromosome inactivation, producing almost exclusive expression of the mutated allele. The G6PD Viangchan variant is characterised by severe enzyme deficiency, but not chronic hemolysis. This study suggests that the metabolic consequences of a combined deficiency of GPI and G6PD might be responsible for a different clinical outcome than predicted for either defect in isolation.     

Increased IgG molecules bound to the surface of red blood cells of patients with sickle cell anemia

We have used the complement-fixing antibody consumption ( CFAC ) test to detect small concentrations of IgG on red blood cells from patients with hemolytic anemias that are not thought to be caused by an immune mechanism. Although patients with hereditary spherocytosis, pyruvate kinase deficiency, and mechanical hemolytic anemias generally had normal concentrations of IgG bound to their red cells (less than 25 molecules IgG per red cell), we found that 39/62 (63%) patients with sickle cell anemia had elevated values. These 39 patients had a mean of 195 and a maximum of 890 molecules of IgG per red cell. None of the patients had been transfused within the previous 90 days, and some had never been transfused. Direct antiglobulin tests were positive in only two instances and autoantibodies were not found in the serum of any patient. However, eluates from the red cells of 6 of 23 patients demonstrated antibody activity against all of a panel of normal red cells by the indirect antiglobulin test. There was no correlation between the number of IgG molecules on patients' red cells and the severity of their anemia, the incidence of painful sickle cell crises, the reticulocyte count, or with blood transfusion history. We conclude that further study of immunohematologic abnormalities in patients with sickle cell anemia is warranted, especially in view of previous reports in this population of patients with red cell autoantibodies, autoimmune hemolytic anemia, hemolytic transfusion reactions without detectable alloantibodies, and an association of some episodes of pain crises with immunologically mediated red cell destruction.     

Red blood cell glucose metabolism in trisomy 10p: possible role of hexokinase in the erythrocyte

Red blood cell glucose metabolism was investigated in a male patient with de novo trisomy 10p. According to previous evidence, when assigning hexokinase gene locus in the 10p11 leads to pter region, a triplex dosage effect of hexokinase activity (HK) was found, while all the other erythrocyte glycolytic enzymes were in the normal values range. Red blood cell glucose utilization was 2.87 mumole/hr/ml RBC as compared to 1.43 in normal controls; the rate of glucose metabolized through the hexose monophosphate shunt (HMPS) was unchanged. Glucose-6- phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and dihydroxyacetone phosphate increased with respect to normal controls, while normal levels of 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and ATP were found. The HK activity increased in all the red blood cell fractions obtained by density gradient ultracentrifugation. However, a small difference in the distribution of cells through the gradient was evident. The experiments reported in this article show that in the red blood cells of patients with trisomy 10p, an increased level of HK leads to higher concentrations of glucose- 6-phosphate and to a faster glucose utilization in the Embden-Meyerhof pathway, while the HMPS rate is unchanged.     

Diagnosis of pyruvate kinase deficiency in a transfusion-dependent patient with severe hemolytic anemia

In a 2-yr old girl a hemolytic anemia was present since birth requiring multiple blood transfusions. Pyruvate kinase deficiency was suspected on the basis of a marginal enzyme activity, but could not be established due to the presence of massive numbers of donor cells in her peripheral blood. However, by density fractionation we succeeded in the isolation of a small fraction of the patient's own cells, in which a severe pyruvate kinase deficiency could be detected. In contrast hexokinase and glucose-6-phosphate dehydrogenase activities were extremely high, which is indicative that a very immature cell population is present in this fraction. In immunofluorescence studies a clear crossreaction was apparent with anti M2-type pyruvate kinase antibodies, whereas only a faint reaction with anti L-type could be detected. Despite the presence of a slight amount of L-type immunoreactive material, the residual activity in the patient's cell fraction could only be attributed to M2-type pyruvate kinase as was shown by cellulose acetate electrophoresis.     

Reticuloendothelial Cell Function in a-Methyldopa-Induced Hemolytic Anemia

Abstract. 2 patients having α-methyldopa-induced hemolytic anemia were followed sequentially using an in vitro assay of autologous monocyte-macrophage activity to determine if their reticuloendothelial system (RES) function was abnormal and thus could be related to the mechanism of lysis. RES function was evaluated while the patients were actively hemolyzing and during remission, following discontinuance of the drug. The results indicated that RES activity is normal in patients having hemolytic anemia due to a-methyldopa administration. Also, following cessation of drug therapy, the patients' IgG-coated red cells interacted significantly for a prolonged period (4–5 months) with autologous or normal allogeneic monocyte-macrophages. This was associated with a concurrent reti-culocytosis and indicates a persistent low-level hemolytic phase throughout this period, even though hemoglobin and hematocrit values remained within the normal ranges. Although levels of IgG sensitizing the patients' red cells were essentially constant during the hemolytic phase and when the patients were in complete remission, significant monocyte-macrophage activity was only evident during the hemolytic period. In an attempt to explain this phenomenon, it is postulated that hemolysis in patients receiving α-methyldopa is related to the interaction of drug with red cell membrane proteins which results in a variably expressed 'altered' antigen which is recognized by 'autoantibody'. The proper expression of the Fc portion of the immunoglobin molecule to result in specific recognition by receptors on monocyte-macrophages depends upon the extent of the antigen alteration by α-methyldopa. If the drug does not result in appropriate antigen alteration, then, although 'autoantibody' may still bind to the red blood cell, its Fc region is not readily recognized by monocyte-macrophages and little or no erythrophagocytosis occurs.     

Case report: hypothyroidism as a possible cause of an acquired reversible hemolytic anemia

The lipid composition of the red cell membrane and plasma was investigated in a patient with hypothyroidism, in whom an acquired hemolytic anemia was reversed after thyroid hormone replacement therapy. Before therapy, most of the plasma lipids were elevated. In the red cell membrane, phosphatidylcholine (PC) and free cholesterol (FC) were increased, and the free cholesterol to phospholipid (FC/PL) ratio was elevated. Erythrocyte sodium transport was also increased, while intracellular sodium and potassium concentrations were normal. After therapy, the derangement of lipid levels and sodium transport activity were normalized with improvement of the hemolytic anemia. The shape of peripheral red cells also returned to normal after treatment. These findings suggest that the derangement of the red cell membrane lipids and plasma lipids derived from hypothyroidism can be a major cause of hemolysis in this patient.     

Anemia and the liver. Hepatobiliary manifestations of anemia

Many types of hemolytic anemia may be associated with liver disease. Liver injury can be caused by the adherence of deformed or hemolyzed erythrocyses to hepatic vascular endothelium. Adhesion of large numbers of hemolyzed red blood cells to hepatic macrophages, or occlusion of hepatic sinusoids by fragmented red cells, can also result in injury of the liver. Thrombosis of the hepatic or portal vein is associated with some types of hemolytic anemia, and can cause severe liner injury. These are some examples of hepatic injury that can be caused by hemolytic anemias. This article discusses some aspects of liver disease that is associated with sickle cell anemia, paroxysmal nocturnal hemoglobinuria, glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, and HELLP syndrome.     

Sulfhemoglobinemia and Acute Hemolytic Anemia with Heinz Bodies Following Contact with a Fungicide-- Zinc Ethylene Bisdithiocarbamate--in a Subject with Glucose-6-Phosphate Dehydrogenase Deficiency and Hypocatalasemia

Sulfhemoglobinemia associated with Heinz body formation and acutehemolytic anemia following contact with a fungicide, zinc ethylene bisdithiocarbamate, is described in a Persian Jew whose red blood cells had low glucose-6-phosphate dehydrogenase activity with low and unstable reducedglutathione and low catalase activity.

The fungicide, similarly to acetylphenylhydrazine, was capable of decreasing in vitro the reduced glutathione of the patient’s red blood cells, as wellas of those of other subjects with the same enzymatic defect.

The sulfhemoglobinemia and the hemolytic anemia are considered to havebeen produced independently by the fungicide, the glucose-6-phosphate dehydrogenase deficiency having played a role only in the latter. The possibilitythat the hypocatalasemia was a factor in rendering the patient’s red bloodcells sensitive to the hemolysis- and sulfhemoglobin-producing action of thefungicide is discussed.

The importance of zinc ethylene bisdithiocarbamate as a sulfhemoglobin-producing and hemolytic agent is stressed, in view of the widespread useof this fungicide.

Submitted on August 3, 1962 Accepted on December 11, 1962     

Atypical familial hemolytic anemia

1. The genetic, clinical and hematologic features of an atypical chronic hemolytic anemia in two siblings of a French Canadian family have been described.

2. The anemia is normocytic, normochromic and not associated with anycharacteristic morphologic abnormality of the red cells.

3. Slight increases in osmotic and incubated mechanical fragility, as well asa more definite increase in aumtohemolysis were found which could not be demonstrated after splenectomy.

4. The survival time of normal red cells was shortened before splenectomy inone patient. Normal red cell survival was demonstrated in both patients aftersplenectomy.

5. The features which differentiate this hemolytic anemia from hereditaryspherocytosis are discussed.

6. French or French Canadian ancestry has been noted in some of the reportedpatients most similar to our own.

7. The association of this type of hemolytic anemia with blood group A hasbeen confirmed in our patients.

8. Splenectomy decreased the severity of the hemolytic process in both patients. This benefit may have resulted from removal of an extracorpuscuarhemolytic mechanism.

Submitted on September 15, 1955 Accepted on October 27, 1955     

Phosphoglycolate phosphatase and 2,3-diphosphoglycerate in red cells of normal and anemic subjects

Red cell phosphoglycolate phosphatase (PGP) and 2,3-diphosphoglycerate (2,3-DPG) were investigated in normal and anemic patients and rabbits. In hemolytic anemia and blood-loss anemia, characterized by a young red cell population, there was an increase in both phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. In aplastic anemia, the phosphoglycolate phosphatase activity was normal, but the 2,3-diphosphoglycerate values were nonetheless increased. Thus, no relationship was found between phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. The lack of correlation between the activity of phosphoglycolate phosphatase and 2,3-DPG levels suggests that modulation of phosphoglycolate phosphatase activity does not control the level of 2,3-DPG in erythrocytes.