The Hereditary Hemolytic Anemias

Symposium on Pediatric Hematology

The Hereditary Hemolytic Anemias Membrane and Enzyme Defects

Denis R. Miller, M.D. *

The hereditary hemolytic anemias are the result of inborn errors of metabolism involving the three main components of the red cell: the membrane, the hemoglobin molecule, or the intracellular enzymes. During the past decade a massive proliferation of knowledge concerning the function and metabolism of the human erythrocyte has resulted in the recognition of defects of membrane structure and function, abnormalities of hemoglobin structure or synthesis, and deficiencies of enzymes, all of which result in shortened erythrocyte survival. The red cell is one of nature's ultimate examples of a harmonious blending of structure and function. The mature, nonreplicating erythrocyte is incapable of synthesizing hemoglobin, membrane protein, or metabolic enzymes required to support its primary role - the delivery of oxygen to the tissues from the lungs and the transport of carbon dioxide from the tissues to the lungs. The cell's metabolic machinery provides the energy required to maintain cation gradients, generate pyridine nucleotides which serve as cofactors to reduce the hemoglobin molecule, protect proteins against oxidative denaturation, and maintain the biconcave shape of the cell during its 120 day lifespan. The purpose of this paper is to discuss those hereditary aberrations of the erythrocyte membrane and enzymes which are responsible for premature cell death. Abnormalities of the hemoglobin molecule are discussed elsewhere in this issue. The interrelationships of abnormal cell structure, metabolism, and function in the pathogenesis of these hereditary hemolytic anemias will be stressed in each of the disorders under consideration.

GENERAL FEATURES OF THE HEREDITARY HEMOLYTIC ANEMIAS As a group, the hereditary hemolytic anemias share many clinical and laboratory features. 14 Clinically these include (1) hyperbilirubinemia "Associate Professor of Pediatrics and Director of Pediatric Hematology, Cornell University Medical College, New York, New York Supported in part by a grant from the U,S. Public Health Service (AM14691-02) and The Children's Blood Foundation, Inc, Pediatric Clinics of North America- Vol. 19, No.4, November 1972

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in the newborn period, occasionally necessitating exchange transfusion, (2) mild scleral icterus, (3) splenomegaly, progressive with age, (4) exacerbation of hemolysis with infection, (5) susceptibility to hypoplastic crises, (6) variable severity of anemia, ranging from mild compensated hemolysis without transfusion requirements to a severe, uncompensated hemolytic process which is transfusion-dependent, and (7) familial incidence. Patients demonstrate laboratory evidence of a hemolytic anemia and commonly have reticulocytosis, erythroid hyperplasia in the marrow, decreased plasma or serum haptoglobin and hemopexin, mild indirect hyperbilirubinemia, and normal hemoglobin electrophoresis. Serologic tests for the presence of abnormal antibodies on the surface of the red cell and for he moly sins or agglutinins in the serum are negative in these patients.

THE RED CELL MEMBRANE-STRUCTURE AND FUNCTION Approximately 50 per cent of the dry weight of the red cell membrane is protein and 44 per cent is lipid, of which three quarters is phospholipid and one quarter is cholesterol.2l, 64, 72 The remaining 6 to 7 per cent is composed of carbohydrates (neutral sugars, hexoseamines, and glycoproteins) which provide the cell with its negative charge (sialic acid) and impart blood group specificityY Some of the membrane proteins may possess enzyme activity, such as transport ATPase, and others, thought to represent about 20 per cent of the total, may have a structural role. 72 Although the mature red cell is incapable of synthesizing lipids, much of the membrane lipid, and particularly cholesterol, exchanges with plasma lipidsY Much more is known about the constituents of the red cell membrane than about the exact organization, arrangement, and conformation of these components which form a lipid-protein complex within it. On the basis of sophisticated electron microscopic studies employing techniques such as freeze-etching or freeze cleaving,92 a number of models have been proposed to incorporate these components into a dynamic and flexible structure capable of performing its many functions, the most important of which are transport, exchange, and diffusion of gases, cations, and metabolites. Membrane deformability and the ability to traverse the microcirculation is dependent upon maintenance of these components of the membrane, a function critically dependent upon a steady source of adenosine triphosphate (ATP), the depletion of which results in a loss of the normal biconcave disc shape with transformation to a sphere, a loss of lipids (fragmentation) and cations (osmotic lysis), increased rigidity (associated with calcium accumulation), and decreased survival,89-91 CATION TRANSPORT. The normal erythrocyte contains a high concentration of potassium (K+) and a low concentration of sodium (Na+). Cations are actively transported or pumped across the red cell membrane against concentration gradients to preserve internal osmotic homeostasis, and to prevent osmotic hemolysis. Energy for this cation "pump"

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THE HEREDITARY HEMOLYTIC ANEMIAS

is derived primarily from ATP, generated by anaerobic glycolysis, and there is evidence that the active sodium-potassium pump of the red cell is linked to the enzyme, ATPase. 57 ,7o Three separate membrane pumps, accounting for 90 to 95 per cent of total Na+ -extrusion, have been characterized. 23 About 70 per cent of cation transport is dependent upon ATPase, which in turn requires magnesium, and is activated by Na+ and K+. The relatively slow rate of cation transport and diffusion is thought to be related to the presence of positively charged pores or channels between the lipoprotein complexes or micelles within the membrane. Normally the erythrocyte actively extrudes (efflux) about 3 millimoles of Na+ per liter of red cells per hour and pumps in (influx) about 2 millimoles of K+ per liter of red cells per hour.95 Approximately 25 per cent of the ATP generated by anaerobic metabolism is utilized for cation pumping and, thus, abnormalities of glucose metabolism and deficiencies of glycolytic enzymes which generate ATP would be expected to result in membrane malfunction and abnormal cation permeability. Cations pass through the red blood cell membranes by two other processes: 95 passive diffusion or "leak," which is in the direction of concentration gradients, and exchange diffusion, in which internal cations are exchanged for similar external cations. These features of cation movement or "flux" are represented in Figure 1. Abnormalities of cation homeostasis caused by inhibition of the cation pump, deterioration of the activity of ATPase, or deficiency in the production of ATP might result in excessive permeability to Na+, accumulation ofNa+ and with it, water, or in excessive loss of K+ and water. The former state would be associated with cellular overhydration and the latter with dehydration. 45

LABORATORY EVALUATION OF MEMBRANE FUNCTION Alterations in the ratio of the surface area of the erythrocyte and its volume, determined by the intracellular osmotically active constituents

K+ [4-5 mM/L]

Na+ [140mM/L]

- - . . . ACTIVE TRANSPORT _ PASSIVE "LEAK" _ EXCHANGE DIFFUSION Figure 1. Cation transport and diffusion in the red cell. Na+ and K+ are actively extruded or pumped in respectively against concentration gradients. Passive diffusion or "leak" occurs in the direction of concentration gradients (K+ out, Na+ in) and exchange diffusion results in the exchange of internal cations for similar external cations.

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will adversely affect cell shape, deformability, resistance to hemolysis in hypotonic solutions of saline, and, eventually, in vivo survival. A number of laboratory determinations are available for evaluating the erythrocyte membrane. Simplest and most rewarding is the microscopic examination of the peripheral blood. Morphologic abnormalities, all considered to represent primary hereditary defects of the red cell membrane will be readily apparent. Representative examples of hereditary spherocytosis, hereditary elliptocytosis (ovalocytosis), and hereditary stomatocytosis are presented in Figure 2. A secondary defect of the red cell membrane, abetalipoproteinemia is also associated with characteristic morphologic abnormalities. Phase contrast microscopy of wet preparations of fresh

Figure 2. Morphologic abnormalities of the erythrocyte associated with membrane defects. A, Hereditary spherocytosis; B, hereditary elliptocytosis; C, hereditary stomatocytosis; D, acan thocytosis in abetalipoproteinemia.

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cells or of cells fixed in glutaraldehyde and the elegant scanning electron microscope30 have yielded much useful additional information. The measurement of osmotic fragility in fresh cells and blood sterilely incubated for 24 hours is extremely useful in evaluating membrane function and integrity.2o Factors which decrease the ratio of surface area: volume, such as excessive permeability to sodium (and water) or loss of membrane lipid, would result in increased spheroidicity and increased osmotic fragility, as occurs in hereditary spherocytosis and in some but not all cases of hereditary elliptocytosis and hereditary stomatocytosis. Hereditary hemolytic anemias associated with decreased osmotic fragility include such disorders as the thalassemia syndromes, sickle cell anemia, and other hemoglobinopathies. The autohemolysis test l4 • 98 is a useful but nonspecific screening test for many inherited intracorpuscular defects. When defibrinated blood is incubated under aseptic conditions for 48 hours at 37° C., with or without nutrient additives (saline, glucose, or ATP at pH 6.8), the degree and correction of hemolysis follow patterns which aid in differentiating some of the hereditary hemolytic anemias. Whereas glucose corrects the increased autohemolysis in certain hereditary hemolytic anemias (Type I pattern of Dacie), glucose fails to correct the abnormality in other types (Type II pattern) which are corrected by ATP. Autohemolysis patterns in various hereditary hemolytic anemias are presented in Table l. Quantification of the content of intracellular cations, rates of cation flux, the composition of membrane phospholipids, the activity of ATPase and determinations of the deformability and filterability of erythrocytes provide further information concerning the structure and function of the erythrocyte membrane.

HEREDITARY DEFECTS OF THE RED CELL MEMBRANE The hereditary disorders associated with abnormalities of the red cell membrane are classified in Table 2. A discussion of the most common types follows.

Characteristic Patterns for Autohemolysis in Various Hereditary Hemolytic Anemias

Table 1.

DEGREE OF HE MOL YSIS

Saline

DIAGNOSIS

Normal red blood cell Hereditary spherocytosis Hereditary elliptocytosis Hereditary stomatocytosis G-6-PD defiCiency

3.5%

marked t moderate t moderate t slight t or normal Pyruvate kinase deficiency moderate to marked t :::May be Type I or normal.

Dacie Pattern

Glucose

ATP

1.0%

1.0%

Normal

normal or slight t normal or slight t normal or slight t normal or slight t

normalort normal normal normal ort

I

moderate to marked i

normal or slight t

IF

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Table 2.

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Classification of the Hereditary Defects of the Red Cell Membrane

Primary Membrane Defects with Specific Morphologic Abnormalities Hereditary spherocytosis Hereditary elliptocytosis Hereditary stomatocytosis with increased osmotic fragility (OF), or high Na+, low K+ with decreased OF, high Na+, low K+ with Rh n "lI Altered Phospholipid Composition Increased lecithin (phosphatidylcholine) Hereditary ATPase deficiency Secondary membrane defects Abetalipoproteinemia

PRIMARY MEMBRANE DEFECTS WITH SPECIFIC MORPHOLOGIC ABNORMALITIES

Hereditary Spherocytosis Hereditary spherocytosis (HS) is the commonest and most studied of the inherited disorders of the red cell membrane, yet the specific defect of the membrane has evaded detection. Characteristically, the disease is inherited as an autosomal dominant trait but in approximately 10 to 20 per cent of the cases,97 there is no familial incidence of the disease and hematologic studies performed in parents of an affected child fail to reveal either the typical morphologic abnormalities or the characteristically increased osmotic fragility and autohemolysis. Atypical cases of hereditary spherocytosis have been reported also, and the possibility exists that these randomly occurring patients represent either spontaneous mutations or heterogeneity of the disorder. A fuller understanding of these unusual cases must await elucidation of the nature of the genetic abnormality. The disease may present in the newborn period with indirect hyperbilirubinemia, reticulocytosis, normoblastemia, and typical spherocytes. 71 Exchange transfusions have been necessary in some affected infants and kernicterus has been reported. 62 It is impossible to differentiate morphologically the spherocytes of hereditary spherocytosis from those associated with ABO incompatibilities, but the presence of a blood group incompatibility, positive Coombs' test, and decreased red cell acetylcholinesterase31 would support the diagnosis of the latter. Infants with hereditary spherocytosis have a variable degree of hemolysis but appear to have their gravest difficulties during the first few months of life when combined bone marrow hypoplasia and brisk hemolysis often necessitate frequent blood transfusions. The hemoglobin ranges between 7 and 10 gm. per 100 ml., and the reticulocyte count between 3 and 15 per cent. The number of spherocytes may vary widely and in some cases the increased osmotic fragility is detectable only after incubation. Splenomegaly may be minimal initially. In other patients, anemia may not be evident until the first year or two of life. A hypoplastic, reticulocytopenic, or aplastic crisis commonly induced by infections

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may precipitate profound pallor, which may be the initial manifesta'tion of the disease. Many severely affected patients appear to be more completely compensated after the first year or two of life when previously required transfusions are no longer necessary. Except for the dangers of hypoplastic crises induced by infection, these children have few difficulties. Splenectomy, the treatment of choice, is performed after the age of 4 or 5 years to obviate the risk of overwhelming infection, especially that caused by the pneumococcus in younger infants and children,16 and before the age of 10 or 12 to decrease the incidence of cholelithiasis, common after puberty. The abbreviated survival of the erythrocyte requires the interaction between an intrinsically defective erythrocyte and a normal spleen. Although glycolytic activity of the hereditary spherocyte is increased and is related to the young cell population under investigation,6o no specific abnormalities of glycolytic enzymes,76 and intermediates 69 have been described. Several investigators 6 • 24, 66 have found that the hereditary spherocyte is hyperpermeable to Na+, but this deviation from nor'mal is now considered to be a secondary and not a primary defect. Studies of the composition of red cell lipids 59 have failed to detect any abnormalities although it has been shown that symmetric loss or fragmentation of whole portions of red cell membrane occurs with incubation. H7 . HH Current concepts of the disorder suggest that a mutant gene produces an abnormality (yet undetermined) of red cell stromal protein which results in rigidity (as measured by the micropipette or filtration)34 of the red cell, predisposing it to entrapment within the splenic pulp where the combined effects of stasis, hypoxia, and acidosis result in fragmentation of the red cell membrane, increased spheroidicity caused by a decreased surface area: volume ratio which further increases red cell rigidity and enhances intrasplenic sequestration. Recent studies 25 have suggested an instability of stromal proteins but attempts to detect abnormalities in the pattern of protein electrophoresis have yielded inconsistent results. 99 The response to splenectomy however is excellent in all patients with the disease. Red cell survival increases to near normal in surgically treated patients and the clinical symptoms and laboratory evidence of hemolysis abate. In our clinic children under 4 or 5 who require splenectomy are placed on continuous prophylactic penicillin following the procedure. Older children are seen promptly for any infection and treated with penicillin until culture reports are available. Although the period of greatest risk to overwhelming infection appears to be within the first year or two after surgery, the later occurrence of significant life threatening infection has been encountered.

Hereditary Elliptocytosis As with hereditary spherocytosis, the specific membrane aberration responsible for the abnormal morphologic features of the erythrocytes in hereditary elliptocytosis has not been discovered. Despite the striking morphologic abnormalities in which up to 90 per cent of the erythrocytes may be elliptocytic (ovalocytic), pencil-shaped or cigar-shaped with smooth surfaces, or occasionally irregularly shaped with crenated, ir-

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regular, or spiculated surfaces, approximately 90 per cent of the patients with this disorder do not have any evidence of hemolytic anemia. 14 The disease is inherited as an autosomal dominant. Examples of double heterozygosity, e.g., hereditary elliptocytosis occurring with thalassemia trait,1 G-6-PD deficiency,53 sickle cell trait,84 and hemoglobin C trait2 have been recorded. Significant hemolytic anemia associated with hereditary elliptocytosis occurs in about 10 per cent of the patients. 2 • 56 The clinical features and the response to splenectomy are similar to those in hereditary spherocytosis. Such complications as severe hyperbilirubinemia necessitating exchange transfusion, reticulocytopenic crises related to infections, and cholelithiasis have been reported in this disorder. Special hematologic investigations may reveal a small percentage of cells with increased osmotic fragility, most marked after incubation, and slightly increased autohemolysis, usually corrected with the addition of glucose or ATP. Erythrocyte survival as measured by 51Cr is modestly shortened, and splenic sequestration of labeled erythrocytes is often demonstrable if surface counting is performed. The pathogenesis of the shape transformation and the shortened erythrocyte survival remains an enigma. Erythrocyte glycolytic activity is normal and commensurate with the age of the red cells, and no defects in energy metabolism, lipid, and protein composition of the membrane have been detectedY

Hereditary Stomatocytosis The stomatocyte is an unusually shaped cell which has a slit-like rather than a circular area of central pallor. Stomatocytes reseJI?ble baskets, cups, or pinch-bottles when viewed with phase contrast microscopy or with the scanning electron microscope after fixation in glutaraldehyde. 39 These cells may be seen in normal blood smears as well as in the peripheral blood of patients with lead poisoning, thalassemia, and acute alcoholism. A number of recent reports 36 • 51. 100 have described a hereditary hemolytic anemia of variable severity characterized by an autosomal dominant mode of inheritance, increased numbers of stomatocytes, reticulocytosis, increased osmotic fragility, and, when measured, a reversal of the usual concentrations of intercellular Na+ and K+, i.e., these cells contained high Na+ and low K+ concentrations. A markedly increased permeability to Na+ and extremely high rates of cation flux were also noted. In a variant of hereditary stomatocytosis40 which has been reported, the cells had decreased osmotic fragility, a modest elevation of intracellular Na+ with slightly decreased intracellular K+, and markedly increased active Na+ efflux with normal K+ influx. As with the other disorders, abnormalities of hemoglobin, intracellular glycolytic enzymes, ATPase, membrane lipid composition, and protein electrophoresis were not detected. Although a membrane and protein defect is suspected to be responsible for the morphologic abnormalities and shortened erythrocyte life span, no primary specific abnormalities of membrane structure have been uncovered to date. Marked variability in the severity of anemia, shortening of red cell survival, the presence or absence of splenic sequestration, and the re-

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sponse to splenectomy have been observed in these heterogeneous states. Whereas some patients have improved after splenectomy, others have had an asymptomatic mild compensated hemolytic anemia which did not require surgical intervention. A propensity to cholelithiasis existed in this latter group of patients.

ABNORMALITIES OF PHOSPHOLIPID COMPOSITION

A hereditary hemolytic anemia occurring in 8 members of a family from the Dominican Republic has been described. 28 The disorder, inherited as an autosomal dominant characteristic, was associated with mild anemia, nonspecific morphologic changes, minimal hyperbilirubinemia, hepatosplenomegaly, decreased osmotic fragility, increased fetal hemoglobin with an otherwise normal electrophoretic pattern of hemoglobin, a type I autohemolysis pattern, normal activity of the glycolytic enzymes, shortened erythrocyte survival, and splenic sequestration. Interestingly the lecithin content of the red cell membrane was increased although the cells contained normal amounts of cholesterol and total phospholipids. No abnormalities in plasma lipids were detected and the relationships between the. lipid disturbance, membrane structure and function, and the hemolytic anemia remain obscure. Abnormalities in cation transport and membrane lipid turnover also exist. Patients have benefitted from splenectomy.

ATPASE DEFICIENCY

Since approximately 70 per cent of active cation transport is dependent upon a Na+-K+ ATPase, a deficiency in the activity of this enzyme was thought to be responsible for the mild to moderate anemia, jaundice, and splenomegaly found in 4 individuals.12' 22 An autosomal dominant mode of inheritance was proposed. Unfortunately, specific, critically important studies of intracellular cation content and flux, essential in characterizing the significance of the enzyme deficiency, were not performed. An autosomal recessive form of ATPase deficiency with increased intracellular sodium has been described in a kindred in which affected individuals did not have hemolysis.

SECONDARY MEMBRANE DEFECTS

Betalipoprotein Deficiency The abnormal composition of phospholipids in the erythrocyte membranes in abetalipoproteinemia is not a primary defect but reflects the inherited deficiency of plasma betalipoproteins, the primary carrier of phospholipid; the plasma deficiency is characterized by acanthocytosis.65 The membranes of these markedly distorted, spiny cells contain decreased lecithin, increased sphingomyelin, and normal or slightly increased amounts of cholesterol and total phospholipids. Patients may

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have a mild hemolytic anemia, with slight reticulocytosis, slightly increased autohemolysis, and normal osmotic fragility.u The exact etiology of the anemia is complex since deficiencies of vitamin B 12 , folic acid, and vitamin E (tocopherol) have been reported in these patients and all may contribute to ineffective erythropoiesis and premature cell death.

HEREDITARY ENZYME DEFICIENCIES OF THE RED CELV 7 ,79

DEFECTS OF THE EMBDEN-MEYERHOF (ANAEROBIC) PATHWAy78

The enzyme reactions of Embden-Meyerhof (EM) or anaerobic pathway account for about 90 per cent of glycolysis within the mature erythrocyte and are illustrated in Figure 3. For each mole of glucose entering the EM pathway, 2 moles of lactate and a net of 2 moles of ATP are produced, since 2 moles of ATP are utilized to phosphorylate glucose in the hexokinase reaction and fructose 6-phosphate in the phosphofructokinase (PFK) reaction. Adenosine triphosphate is generated at the phosphoglycerokinase (PGK) and pyruvate kinase (PK) steps. In addition to serving as the energy source for ATPase-dependent cation transport, ATP is also important for the maintenance of the normal biconcave disc shape of the erythrocyte, membrane lipid, critical hemolytic volume, and membrane protein. Finally, ATP is utilized for the synthesis of purine and pyridine nucleotides via the salvage pathway from adenine and from small precursors respectively. Metabolic defects associated with perturbations of glycolysis caused by inherited deficiencies of glycolytic enzymes often result in decreased ATP synthesis or increased ATP utilization. The common final pathway of shortened erythrocyte survival in these deficiency diseases may be related to the formation of rigid, nondeformable erythrocytes which would be trapped in the microcirculation of the reticuloendothelial system,93 particularly the spleen, liver, and bone marrow. Stasis, acidosis, and hypoxia with inhibition of glycolytic activity and progression of rigidity would enhance the premature destruction of these intrinsically abnormal cells whose metabolic handicaps are incapable of tolerating further embarrassment or stress. The presence of occasional spherocytes in the peripheral blood of patients with enzymopenic hemolytic anemias (e.g., glucosephosphate isomerase,50 PK after splenectomy)44 emphasizes the misnomer "nonspherocytic hemolytic anemias" which had been used to characterize these metabolic disorders prior to the definition of specific enzyme deficiencies. Another important product of anaerobic metabolism is NADH (DPNH). The primary pathway for reduction of methemoglobin formed within the erythrocyte is catalyzed by an NADH-dependent methemoglobin reductase. 61 NADH is generated at the glyceraldehyde phosphate dehydrogenase (G3PD) step. Congenital deficiency of NADH-dependent methemoglobin reductase results not in hemolytic anemia but rather in

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THE HEREDITARY HEMOLYTIC ANEMIAS

y-Glut-Cyst + Gly

Glucose Arp

...J Mg* 'G'Luc'osf:s:

.....:rp

DE~~g~~~mSE

AOP.-t

.... AOP

1

GLUTATHIoNE"

1'~'~~!'~'~:':~'~~'

~i~;"ii';"'~~::CZ~I~w y _y~,O, .................................

Arp

~

\

PHOSPHOGLUCONATE DEHYDROGENASE

H \

GLUTATHIONE REDUCTASE

GLUTATHIONE PEROXIDASE

.,.~~~"'''''' ""F lo6: \;~;=f~- A GSS~X~,o o

;:.:.::::;~~ )--ROSOP G-3-P

Pi~

GLYCERALDEHYDE.3-PHOSPHATE DEHYDROGENASE

1,3-0PG

.•••••••••••••••••••••••••••••••••••••• AOP

.~!!~~!.!!~~.~!.~.~~~:~···~~~~·~~rp

...f

.-t

HAD

.... HAOH

/

~r.

J-~ Mg H

'OIPHOSPHOGLYCERiiMU'TASE'

'\ -'...................\ ............. ..

2.3-0PG Pi)

3-PG+,--------~\---J·

6

~-DPG

PHOSPHOGLYCEROMUTASE

DIPHOSPHOGLYCERATE PHOSPHATAsr

2-PG PHOSPHOPYRUVATE

HYDRATASE

I

Mg ....

PEP

AOP

Arp

,~"" ~'''.'''~"

~

MgH

4 K'

P"f= Loctote

Figure 3. Enzyme reactions of the Embden-Meyerhof and hexose monophosphate pathways of metabolism. Documented hereditary enzyme deficiency diseases are indicated by the enclosing dotted lines. Seven defects of the Embden-Meyerhof pathway and 4 of the hexose monophosphate pathway shunt have been described. (From Valentine, W. N.: Sem. Hemat., 8:348, 1971, by permission.)

cyanosis. In some patients severe mental retardation has been an associated finding. A deficiency of any of the glycolytic enzymes may exert its effects not only directly by the failure of generation of a vital cofactor but also by feed-back inhibition of other metabolic steps. For example, erythrocyte PK deficiency is associated with increased intracellular 2,3-diphosphoglycerate (2,3-DPG)61 and decreased NAD (DPN).19 Increased 2,3-DPG inhibits the hexokinase reaction,1O the first step of the EM pathway; decreased NAD decreases the activity of G3PD and results in an accumulation of hexose sugars above this step.63 An erythrocyte enzyme deficiency disease may be caused by the decreased production of an enzyme (quantitative defect) or to the produc-

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tion of a functionally abnormal enzyme (qualitative defect). Advances in methods of enzyme analysis have resulted in the identification of deficiencies of 7 enzymes in the EM pathway: pyruvate kinase, hexokinase, glucosephosphate isomerase, phosphofructokinase, triosephosphate isomerase, phosphoglycerate kinase, and diphosphoglyceromutase.78 During the past few years, the marked heterogeneity of enzyme deficiency diseases has become apparent. For example at least 69 different variants of G-6-PD deficiency7 and 8 variants of PK deficiency have been described. 73 The identification of these variants which often produce similar clinical findings requires special studies, including enzyme kinetics, purification, and fingerprinting. An important alternate metabolic pathway of glycolysis is the 2,3DPG or Rapoport-Luebering shunt in which 2,3-DPG is synthesized from 1,3-DPG by the enzyme 2,3-DPG mutase and catabolized to 3-PG by 2,3DPG phosphatase.32 If the flow of glycolysis is through the 2,3-DPG cycle, ATP production at the PGK step would be bypassed and no net synthesis of ATP would occur. Of all the intracellular glycolytic intermediates, 2,3DPG is in highest concentration and by combining with deoxyhemoglobin decreases the affinity of hemoglobin for oxygen and shifts the oxygen association curve to the right. 5 The physiologic importance of 2,3-DPG is discussed elsewhere in this symposium. Pyruvate Kinase Deficiency Pyruvate kinase deficiency73-75.83 has been reported in over 135 patients and is the first described, most common, and best defined enzyme abnormality of the EM pathway. Pyruvate kinase converts phosphoenolpyruvate to pyruvate, a reaction in which ADP is phosphorylated to ATP. Thus, a perturbation of enzyme activity at this step results in decreased ATP generation, and a breakdown of normal cation gradients, K+ loss, increased cell rigidity, and premature destruction. The disease is inherited as an autosomal recessive characteristic with asymptomatic parents possessing about 50 per cent of normal activity. Affected children have about 10 to 25 per cent of normal red blood cell PK activity, although some patients have activity which falls in the heterozygous or normal range. Many patients are of Northern European ancestry and a particularly high incidence among the Amish has been reported. There is a wide variability in the severity of hemolysis of affected children who have the typical manifestations of chronic hemolytic anemia. At one end of the spectrum severe neonatal jaundice occurs and continuous transfusion therapy is required, whereas at the other end the diagnosis is delayed until adulthood because of the mild symptoms and the compensated state of the hemolytic process. Growth and development are often delayed and the bony changes characteristic of chronic hemolytic anemias may be seen. Most patient's cells demonstrate a type II pattern of autohemolysis, whereas some may have a normal or type I pattern. Polychromatophilia, anisocytosis and poikilocytosis are usual but nonspecific morphologic abnormalities. Metabolic studies often reveal (1) overall glycolytic activity which is not commensurate with the degree of reticulocytosis, (2) increased erythrocyte 2,3-DPG, (3) decreased oxygen affinity15 (increased p50 O2 ), and (4) decreased and unstable levels of ATP. Erythrocyte survival of 51Cr-labelled cells is usually de-

THE HEREDITARY HEMOLYTIC ANEMIAS

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creased and a variable pattern of organ sequestration has been noted in several studies. 43 . 73 The early appearance of radioactivity over the spleen and the shortened survival of labelled reticulocytes suggested a preferential destruction of these young cells. It has been proposed that the hypoxia of the spleen is incapable of supporting reticulocyte metabolism, in which the Krebs tricarboxylic cycle and oxidative phosphorylation are the main mechanisms of ATP production.38 The marked variability of the clinical and laboratory findings may be explained partially by genetic polymorphism and molecular heterogeneity.54 A number of variants with both high and low Michaelis constants for PEP (Km PEP) and with or without activation by the allosteric effector fructose diphosphate42 . 73 have been defined recently, and thus, full definition of the deficiency may require kinetic studies. The response to splenectomy, unlike that in hereditary spherocytosis, has not resulted in a cessation of hemolysis but many patients have required fewer transfusions and in some this requirement has been eliminated. Increased packed cell volume, marked reticulocytosis (up to 90 per cent), and improved exercise tolerance have been noted after the procedure. Hexokinase Deficiency27. 78. 82 The initial reaction of the EM pathway is catalyzed by hexokinase, the activity of which is the lowest of all of the glycolytic enzymes. Hexokinase, like PK, G-6-PD, and glutamic-oxaloacetic transaminase, is an exquisitely age-dependent enzyme, its activity declining and inversely correlating with progressive senescence of the red cell. Hereditary hemolytic anemia with hexokinase deficiency has been described in 5 patients. The activity of hexokinase varied between normal and 50 per cent of normal but was significantly reduced when related to the activity of the enzyme in populations of cells with similar reticulocytosis. Although large scale family studies are incomplete, the disease appears to be inherited as an autosomal recessive characteristic. The degree of hemolysis varies from severe to mild; the autohemolysis pattern is either type I or normal. A decreased concentration of 2,3-DPG was associated with increased oxygen affinity and a leftward shift of the oxygen dissociation curve 15 (decreased p50 O2), Kinetic and electrophoretic studies have provided evidence for genetic polymorphism. A partial benefit to splenectomy has been observed. German investigators37 have detected hexokinase deficiency in the erythrocytes, leukocytes, and platelets of 3 patients with Fanconi's aplastic anemia and N echeles48 has reported a deficiency of leukocyte hexokinase in his patients with the red cell deficiency. However, none of the patients with hexokinase deficiency hemolytic anemia had the congenital malformations characteristic of Fanconi's pancytopenia. We have been unable to detect a deficiency of hexokinase in the red cells of patients with Fanconi's anemia followed in our clinic.

1

Glucosephosphate Isomerase Deficiency4 A moderately severe, uncompensated hereditary hemolytic anemia _ _r_eSUlting from glucosephosphate isomerase (GPI) deficiency has been

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reported in 11 patients in whom the leukocytes were also deficient. No increased susceptibility to infection was observed. A type I autohemolysis pattern, autosomal recessive inheritance, and partial benefit from splenectomy have been observed. Electrophoretic differences but normal enzyme kinetics were detected. Glucosephosphate isomerase interconverts glucose-6-phosphate and fructose-6-phosphate, a re-entry point of metabolism (see Fig. 2) from the pentose phosphate shunt. The exact mechanism of hemolysis is unknown but these cells are unable to recycle glucose through the pentose shunt. Phosphofructokinase Deficiency77 Two variants of PFK deficiency hemolytic anemia have been described. The first, inherited as an autosomal recessive trait, was associated with a very mild hemolytic anemia and a myopathy (Type III glycogen storage disease) with myoglobinuria. 77 A deficiency of PFK in skeletal muscle and red cells was demonstrated. In the second variant,86 the PFK deficit was restricted to the red cells and the inheritance pattern appeared to be linked to the X-chromosome. PFK is an important rate limiting control point in glycolysis and is extremely sensitive to pH and ATP concentration. A deficiency of the enzyme could severely handicap glycolytic activity. Triosephosphate Isomerase Deficiency67 In addition to a severe hemolytic anemia, patients with this disorder have a progressive debilitating neuromuscular disease, and some have died of sudden cardiac arrest. The enzyme deficiency occurs in erythrocytes, leukocytes, skeletal muscle, serum, and spinal fluid, and death may occur in early childhood. An autosomal recessive mode of inheritance has been proposed and chromosome 5 is thought to be associated with the enzyme. A toxic or inhibitory effect of dihydroxyacetone phosphate may be operative, but the pathogenesis of hemolysis is unknown, since despite the metabolic block at the interconversion of G3P-dihydroxyacetone phosphate, glycolysis proceeds at a normal rate. Phosphoglycerate Kinase Deficiency A deficiency of phosphoglycerate kinase (PGK) has been reported in males with markedly decreased activity and in females with activity presumed to be in the heterozygous range. Valentine and coworkers 81 have suggested X-chromosome linkage on the basis of extensive studies in a Chinese kindred. These patients had a moderately severe hemolytic anemia as well as mental retardation and a behavioral disorder. Since PFK is an ATP-generating step, an enzyme deficiency would interfere with ATP formation which may be related to premature cell death. Leukocytes deficient in the enzyme exhibited decreased bactericidal activity.:; Diphosphoglyceromutase Deficiency 68 This enzyme converts 1,3-DPG to 2,3-DPG. European investigators have described a moderately severe hemolytic anemia associated with

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slight reticulocytosis, decreased 2,3-DPG and autosomal dominant or recessive inheritance. Evidence for the deficiency in some patients was indirect, as the definitive assay system for 2,3-DPG mutase is much more complex than those used for quantifying other glycolytic enzymes. Although not reported. a low p50" (increased oxygen affinity) would be expected in these cells with decreased 2,3-DPG.

Uncommon Glycolytic Enzyme Defects Single case reports of hemolytic anemia associated with a deficiency of glyceraldehyde-3-phosphate dehydrogenase52 and 2,3-DPG phosphatase 26 have been recorded but further confirmatory and more definitive studies are required. Spherocytes were associated with the latter syndrome. Valentine and co-workers80 described a hemolytic anemia in which a high ATP content and decreased activity of ribose phosphate pyrophosphokinase were found. This enzyme is important in increasing the total pool of adenine nuc1eotides (ATP,ADP,AMP) through the salvage pathway. The exact mechanism of hemolysis and the genetic pattern of inheritance remains to be elucidated. Two other hereditary hemolytic anemias have been described with abnormalities of ATP metabolism. The first, dominantly transmitted, is characterized by low red cell ATP, but no other enzyme defect has been detected. 55 The second is associated with adenylate kinase deficiency which converts 2 moles of ADP to ATP and AMP.78

DEFECTS OF THE HEXOSE MONOPHOSPHATE SHUNT 7

The primary functions of the hexose monophosphate shunt (HMP) are the generation of NADPH and the reduction of oxidized glutathione (GSH). Although only 10 per cent of glycolytic activity of the red cell flows through the HMP, congenital deficiencies of key enzymes (1) directly involved in the pathway (G6PD, 6PGD), (2) concerned with the synthesis, and reduction of GSH (GSH synthetase, GSH reductase) or (3) active in detoxifying hydrogen peroxide (GSH peroxidase) result in significant hemolytic anemia. These main enzyme reactions are illustrated in Figure 3. Certain oxidant drugs, infections, acidosis, other diseases, and the endogenous production of hydrogen peroxide itself may induce hemolysis in patients with deficiencies of enzymes of the HMP. A continuous supply of GSH must be made available to protect sulfhydryl groups of hemoglobin, membrane proteins, and other intracellular enzymes against oxidative denaturation. The primary denaturant, hydrogen peroxide, is detoxified enzymatically to innocuous water by GSH peroxidase, a step in which GSH is converted to oxidized glutathione (GSSG). GSSG is reduced to GSH by GSH reductase and its cofactor NADPH, in turn generated by G6PD and 6PGD. The inadequacy of any of these reactions results in the formation of mixed disulfide linkage involving GSH and globin chains of hemoglobin and eventually denatured, precipitated Heinz bodies. This final common pathway, the formation of Heinz bodies, leads to entrapment in the microcirculation of the spleen (as well as the liver and bone marrow), where Heinz bodies are "pitted out" by phagocy-

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Figure 4. Electron micrograph showing Heinz body-containing erythrocyte traversing the microcirculation of the spleen. A dense Heinz-body in the tail of the erythrocyte is surrounded by a phagocytic reticuloendothelial cell which contains other Heinz-bodies. (With the permission of R. I. Weed, M.D.)

tic reticuloendothelial cells within the splenic cords (Fig. 4), further increasing red cell rigidity, enhancing spheroidicity, decreasing deformability, altering membrane function, and predisposing these cells to an early grave. The presence of microspherocytes in the peripheral blood smears of these patients makes the expression "nonspherocytic hemolytic anemia" untenable, and, as a meaningless term, it too should be put to rest. Hereditary hemolytic anemia with Heinz body formation embraces not only those enzyme deficiencies of the pentose phosphate shunt, but also the unstable hemoglobin hemolytic anemias and the thalassemia syndromes in which the imbalance in chain production leads to precipitation of globin chains as Heinz bodies.

Glucose-6-Phosphate Dehydrogenase Deficiency The series of studies relating primaquin-sensitivity to a deficiency of red cell G-6-PD is clearly one of the most exciting research sagas in hematology. Probably the most common inborn error of metabolism in man, G-6-PD was the first erythrocyte enzyme deficiency discovered and today extensive work on the genetics, biochemistry, and molecular pathology of the disorder make it the best understood and most thoroughly studied of the enzymopathies. As of October 1971,69 variants of G-6-PD7 have been documented with distinctive electrophoretic, kinetic, and physicochemical characteristics, but not all are associated with either hereditary hemolytic anemia or deficient enzyme activity. A single amino

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acid substitution (asparagine to aspartic acid) distinguished the B+ enzyme found in Caucasians from the A+ enzyme which occurs in 18 per cent of Blacks. 96 Every example of the deficiency however is characterized by X-chromosome linkage, affecting male offspring of heterozygous mothers. The disorder is found in 10 to 15 per cent of Black American males, in whom clinically significant hemolysis occurs after ingestion of certain antipyretics (e.g., acetanilid), analgesics, antimalarials (e.g., 8amino-quinolines), sulfonamides, sulfones, nitrofurans, and naphthalene. The disease is also common in the Mediterranean littoral (among Sardinians, Greeks, and Sephardic Jews) and in the Orient. The high incidence of the gene in these areas is thought to be related to genetic polymorphism in which G-6-PD deficiency protects against fatal falciparum malaria. Recent studies have related this protection to the presence of the A- variant. HH Rarely, in Caucasians, G-6-PD deficiency causes a chronic congenital hemolytic anemia which is unrelated to drug ingestion but may be associated with neonatal hyperbilirubinemia and a mild to moderate hemolytic anemia which rarely requires transfusion therapy.8 In these children, the enzyme may be deficient in other cells, including erythrocytes and platelets. Acute hemolytic episodes following the ingestion of fava beans occurs in a few G-6-PD-deficient Caucasians and Orientals but never in Negroes with the A-deficiency. The enzyme deficiency is the commonest cause of neonatal jaundice and kernicterus in certain areas of Greece!7 but among deficient Blacks, only premature infants appear to have significant hyperbilirubinemia.!8 Since G-6-PD is an age-dependent enzyme, with the activity decreasing with maturity, the older cells are preferentially destroyed. Two to 4 days after drug ingestion, symptoms of acute hemolysis develop, with hemoglobinuria, methemalbuminemia, reticulocytosis, and indirect bilirubinemia. Despite continued drug administration, a plateau or equilibrium phase is achieved, during which time the reticulocyte count and hemoglobin and hematocrit levels return to normal. A second wave of acute hemolysis will occur when the mean cell age of the cohort of cells produced after the first episode of hemolysis reaches the prehemolytic range. Several simple screening tests or a quantitative spectrophotometric assay are available to demonstrate a deficiency of the enzyme. The cells of a deficient individual contain 0 to 26 per cent of normal enzyme activity. Immediately after an acute hemolytic episode, values may fall in the heterozygous or low normal range, but when this activity is compared to that in a population of cells with an equivalent reticulocytosis, the deficiency will be apparent. The presence of Heinz bodies (visualized with supravital dyes such as cresyl violet) and GSH instability are other distinguishing laboratory features. There is no specific treatment for this disorder. Potentially hemolytic drugs should be avoided and preventive screening should be performed in susceptible populations before embarking in therapeutic programs entailing these agents. Splenectomy has rarely, if ever, been beneficial in the rare Caucasian patient with chronic congenital hemolytic anemia caused by G-6-PD deficiency.

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6-Phosphogluconic Dehydrogenase Deficiency35 This extremely rare condition has been reported in 2 patients with hereditary hemolytic anemia, but family, clinical, and biochemical studies are sketchy and the disorder remains incompletely defined.

DISORDERS OF GSH SYNTHESIS, PEROXIDATION, AND REDUCTION

GSH Synthetase Deficiency Glutathione is synthesized in a two-step procedure: first, glutamic acid and cysteine form glutamyl-cysteine, catalyzed by glutamyl-cysteine synthetase,':' and secondly, glutamyl-cysteine is linked to glutamic acid to f-orm GSH, a reaction catalyzed by GSH synthetase. A deficiency of GSH synthetase, associated with markedly decreased intracellular GSH and a mild compensated hereditary hemolytic anemia, and inherited as an autosomal recessive trait has been reported in European58 and American familiesY Hemolysis is exacerbated by oxidant drugs or fava beans. GSH Reductase Deficiency7, 85 A drug-induced congenital hemolytic anemia with GSH reductase deficiency was first reported in 1962. A partial deficiency of the enzyme has been reported in association with diverse hematologic abnormalities including pancytopenia, factor IX deficiency, hemoglobin C disease, and leukemia. Non-hematologic abnormalities such as neurologic disorders and Gaucher's disease have also been described with GSH reductase deficiency. Recent studies have demonstrated that the variability in clinical and laboratory features of the deficiency may be related to a riboflavin deficiency rather than to a congenital deficiency of GSH reductase. 7 Riboflavin binds to the enzyme and increases its activity. A number of patients, presumed to have hereditary GSH reductase deficiency, have been restudied after periods of adequate riboflavin intake and were found to have normal enzyme activity. The in vivo survival of GSH reductasedeficient cells is normal and mild GSH reductase deficiency has no adverse effect upon the activity of the hexose monophosphate shunt. Thus, in most cases, it appears that GSH reductase deficiency is caused by dietary rather than genetic factors. GSH Peroxidase Deficiency7, 46, 47, 49 The activity of GSH peroxidase is physiologically low in normal newborn infants. The increased susceptibility of the newborn infant's red cells to oxidant stress may be related to this maturational deficiency, although a significant correlation was not demonstrated between bilirubinemia and GSH peroxidase activity.94 Kinetic studies of the enzyme may provide an explanation for the decreased activity found in the newborn. Normal adult levels are not achieved until about 6 months of age. A deficiency of this enzyme combined with vitamin E deficiency and sup"Recently, a defiCiency of y-glutamyl-cysteine synthetase associated with a compensated hemolytic anemia and decreased intracellular GSH has been described in two siblings."

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plementary iron intake, may be responsible for the hemolytic anemia observed in premature infants between 6 and 10 weeks of age. GSH peroxidase deficiency has been reported in newborn infants with hemolytic disease46 and in older children and adults with chronic mild to moderate drug-induced hemolytic anemia. 47 ,49 Although extensive genetic studies are still required, the disorder appears to be inherited as an autosomal recessive characteristic. Homozygotes and heterozygotes may have hemolytic anemia. The presence of Heinz bodies in the peripheral blood, normal GSH content, and enzyme activity ranging from 30 to 89 per cent of normal are other distinguishing laboratory features of this recently described disorder.

SUMMARY The hereditary hemolytic anemias associated with defects of membrane structure and function and with deficiencies of critical glycolytic enzymes produce similar clinical syndromes and shortened erythrocyte survival. With certain disorders, particularly those involving the red cell membrane, the specific defect has not been elucidated but a number of secondary abnormalities affecting the ratio of surface area: volume, cation transport, and cell deformability are felt to be important in the premature destruction of the red cell. Specific enzyme deficiency diseases may be caused by the production of a structurally-abnormal protein with abnormal stability, by the inadequate production of protein of normal structure or by the production of protein possessing aberrant kinetics or substrate affinity. Whereas in certain enzyme deficiency diseases only the red cell is affected, in others, other cells and tissues may share the deficit, and increased susceptibility to infection, muscle disease, or mental retardation may be associated findings. Energy production from glycolysis, in the form of ATP, which is required for the normal maintenance of red cell biconcavity, cation homeostasis, "membrane integrity," and other functions, may be relatively or absolutely deficient in the red cells of patients with enzyme deficiency diseases. There appears to be abundant evidence that the genetically determined defect is the primary cause of hemolysis in affected patients. The 12 documented enzymopathies involving the EM and HMP pathways of glycolysis have been reviewed and the interrelationships between abnormal cell metabolism and cell function have been stressed. These abnormalities of abnormal membrane and glycolytic function have provided us with a greater understanding of normal erythrocyte metabolism and the mechanisms of cell destruction. With the vast proliferation of knowledge in this field during the past 10 years, little imagination is required to predict that continued research in the next decade will yield a fuller understanding of the biochemistry, pathophysiology, genetics, and treatment of these inherited molecular disorders and undoubtedly unravel new defects and variants of previously described abnormalities. The documentation of almost 70 varieties of G-6-PD attests

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to this prognostication for future development in the field of red cell metabolism. Shakespeare has written "What a piece of work is man!" The red cell, the "small round globule", described by Leeuwenhoek nearly 300 years ago, is an integral part of the human organism and aptly satisfies the great bard's praise.

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25. Jacob, H. S., Ruby, A., Overland, E. S., and Mazia, D.: A protein abnormality in red cell membranes of hereditary spherocytosis. J. Clin. Invest., 50:1800, 1971. 26. Jacobasch, G., Syllm-Rapoport, E., Rorgas, H., and Rapoport, S.: 2,3-PGase-mangel als mogliche ursache erhohten ATP-Gehaltes. Clin. Chim. Acta, 10:477,1964. 27. Jaffe, E. R: Hereditary hemolytic disorders and enzymatic deficiencies of human erythrocytes. Blood, 35:116,1970. 28. Jaffe, E. R, and Gottfried, E. L.: Hereditary non spherocytic hemolytic disease associated with an altered phospholipid composition of the erythrocytes. J. Clin. Invest., 47:1375, 1968. 29. Jaffe, E. R, and Hsieh, H. S.: DPNH-methemoglobin reductase deficiency and hereditary methemoglobinemia. Sem. Hemat., 8:417, 1971. 30. Jensen, W. N., and Lessin, L. S.: Membrane alterations associated with hemoglobinopathies. Sem. Hemat., 7:409, 1970. 3l. Kaplan, E., Herz, F., and Hsu, K. S.: Erythrocyte acetylcholinestrase activity in ABO hemolytic disease of newborn. Pediatrics, 33:205, 1964. 32. Keitt, A. S.: Pyruvate kinase deficiency and related disorders of red cell glycolysis. Amer. J. Med., 41 :762, 1966. 33. Konrad, P. N., Richards, F. II, Valentine, W. N., and Paglia, D. E.: y-glutamyl-cysteine synthetase deficiency. A cause of hereditary hemolytic anemia. New Eng. J. Med., 286:557, 1972. 34. La CelIe, P. L.: Alteration of membrane deformability in hemolytic anemias. Sem. Hemat., 7:355, 1970. 35. Lausecker, C., Heidt, P., Fischer, D., et a!.: Anemie hemolytique constitutionnelle avec deficit en 6-phospho-gluconate-dehydrogenase. Arch. Fran<;. Paediat. 22:789, 1965. 36. Lock, S. P., Smith, R. S., and Hardisty, R. M.: Stomatocytosis: a hereditary red cell anomaly associated with haemolytic anaemia. Brit. J. Haemat., 1 :303, 1961. 37. Lohr, G. W., Waller, H. D., Anschutz, F., and Knoop, A.: Biochemische defekte in die blutzellen bei familiarer panomyelopathie. (Typ Fanconi). Humangenetik, 1 :383, 1965. 38. Mentzer, W. C., Jr., Baehner, R L., Schmidt-Schoenbein, H., and Nathan, D. G.: Selective reticulocyte destruction in erythrocyte pyruvate kinase deficiency. J. Clin. Invest., 50:688, 1971. 39. Miller, D. R, and Bessis, M.: Unpublished observations. 40. Miller, D. R, Rickles, F. R, Lichtman, M. A., et a!.: A new variant of hereditary hemolytic anemia with stomatocytosis and erythrocyte cation abnormality. Blood, 38: 184, 1971. 41. Mohler, D. N., Majerus, P. W., Minnich, V., et al.: Glutathione synthetase deficiency as a cause of hereditary hemolytic disease. New Eng. J. Med., 283:1253,1970. 42. Munro, G. F., and Miller, D. R: Mechanism of fructose diphosphate activation of a mutant pyruvate kinase from human red cells. Biochim. Biophys. Acta, 206:87,1970. 43. Nathan, D. G., Oski, F. A., Miller, D. R, and Gardner, F. H.: Life-span and organ sequestration of the red cells in pyruvate kinase deficiency. New Eng. J. Med., 278:73,1968. 44. Nathan, D. G., Oski, F. A., Sidel, V. W., et al.: Studies of erythrocyte spicule formation in haemolytic anaemia. Brit. J. Haemat., 12: 385, 1966. 45. Nathan, D. G., and Shohet, S. B.: Erythrocyte ion transport defects and hemolytic anemia: "hydrocytosis and dessicytosis." Sem. Hemat., 7:381, 1970. 46. Necheles, T. F., Boles, T. A., and Allen, D. M.: Erythrocyte glutathione-peroxidase deficiency and hemolytic disease of the newborn infant. J. Pediat., 72:319, 1968. 47. Necheles, T. F., Maldonado, N., Barquet-Chedrak, A., and Allen, D. M.: Homozygous erythrocyte glutathione-peroxidase deficiency: Clinical and biochemical studies. Blood, 33:164,1969. 48. Necheles, T. F., Rai, U. S., and Cameron, D.: Congenital non spherocytic hemolytic anemia associated with an unusual hexokinase abnormality. J. Lab. Clin. Med., 76:593, 1970. 49. Necheles, T. F., Steinberg, M. H., and Cameron, D.: Erythrocyte glutathione-peroxidase deficiency. Brit. J. Haemat., 19:605,1970. 50. Oski, F., and Fuller, E.: Glucosephosphate isomerase (GPI) deficiency associated with abnormal osmotic fragility and spherocytes. Clin. Res., 19:427,1971. 51. Oski, F. A., Naiman, J. L., Blum, S. F., et al.: Congenital hemolytic anemia with highsodium, low-potassium red cells. Studies of three generations of a family with a new variant. New Eng. J. Med., 280:909,1969. 52. Oski, F., and Whaun, J.: Hemolytic anemia and red cell glyceraldehyde-3-phosphate dehydrogenase (G-3-PD) deficiency. Clin. Res., 17:601, 1969. 53. Ozer, L., and Mills, G. C.: Elliptocytosis with haemolytic anaemia. Brit. J. Haemat., 10:468, 1964. 54. Paglia, D. E., Valentine, W. N., Baughan, M. A., et a!.: An inherited molecular lesion of erythrocyte pyruvate kinase. Identification of a kinetically aberrant enzyme associated with premature hemolysis. J. Clin. Invest., 47:1929,1968. 55. Paglia, D. E., Valentine, W. N., Tartaglia, A. P., and Konrad, P. N.: Adenine nucleotide reductions associated with a dominantly transmitted form of nonspherocytic hemolytic anemia. Blood, 36:837,1970.

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56. Pearson, H. A.: The genetic basis of hereditary elliptocytosis with hemolysis. Blood, 32:972, 1968. 57. Post, R. L., Merritt, C. R., Kinselving, C. R., and Albright, C. D.: Membrane adenosine triphosphatase as a participant in the active transport of sodium and potassium in the human erythrocyte, J. BioL Chem., 235:1796,1960. 58. Prins, H. K., Oort, M., Loos, J. A., et aL: Congenital nonspherocytic hemolytic anemia associated with glutathione deficiency of the erythrocytes. Blood, 27:145,1966. 59. Reed, C. F., and Swisher, S. N.: Erythrocyte lipid loss in hereditary spherocytosis. J. Clin. Invest., 45:777,1966. 60. Reed, C. F., and Young, L. E.: Erythrocyte energy metabolism in hereditary spherocytosis. J. Clin. Invest., 46:1196,1967. 61. Robinson, M. A., Loder, P. B., and de Gruchy, G. C.: Red-cell metabolism in nonspherocytic congenital haemolytic anaemia. Brit. J. Haemat., 7:327,1961. 62. Roddy, R.: Clinical Conferences at St. Christopher's Hospital for Children. J. Pediat., 44:213,1954. 63. Rose, I. A., and Warms, J. V. B.: Control of glycolysis in human red blood cell. J. BioL Chem., 241 :4848, 1968. 64. Rosenberg, S. A., and Guidotti, G.: The proteins of the erythrocyte membrane: Structure and arrangement in the membrane. In Jamieson, G. A., and Greenwalt, T. J., eds.: Red Cell Membrane Structure and Function. Philadelphia, J. B. Lippincott Co., 1969. 65. Salt, H. B., Wolff, O. H., Lloyd, J. K., Fosbrooke, A. S., Cameron, A. H., and Hubble, D. V.: On having no beta-lipoprotein, a syndrome comprising a-beta-lipoproteinemia, acanthocytosis and steatorrhea. Lancet, 2:325, 1960. 66. Selwyn, J. G., and Dacie, J. V.: Autohemolysis and other changes resulting from the incubation in vitro of red cells from patients with congenital hemolytiC anemias. Blood, 9:414, 1954. 67. Schneider, A. S., Valentine, W. N., Hattori, M., and Heins, H. L., Jr.: Hereditary hemolytic anemia with triosephosphate isomerase deficiency. New Eng. J. Med., 272:229,1965. 68. Schroter, W.: 2,3-Diphosphoglyceratstoffwechsel und 2,3-diphosphoglyceratmutaseMangel in Erythrozyten. Blut, 20:1,1970. 69. Shafer, A .. W.: The phosphorylated carbohydrate intermediates from erythrocytes in hereditary spherocytosis. Blood, 23 :417, 1964. 70. Skou, J. C.: Enzymatic basis for active transport of Na+ and K+ across cell membranes, PhysioL Rev., 45:596,1965. 71. Stamey, C. C., and Diamond, L. K.: Congenital hemolytiC anemia in the newborn. Amer. J. Dis. Child., 94:616, 1957. 72. Sweeley, C. C., and Dawson, G.: Lipids of the erythrocyte. In Jamieson, G. A., and Greenwalt, T. J., eds.: Red Cell Membrane Structure and Function. Philadelphia, J. B. Lippincott Co., 1969. 73. Tanaka, K. R., and Paglia, D. E.: Pyruvate kinase deficiency. Sem. Hemat., 8:367,1971. 74. Tanaka, K. R., and Valentine, W. N.: Pyruvate kinase deficiency. In Beutler, E., ed.: Hereditary Disorders of Erythrocyte Metabolism. New York, Grune & Stratton, 1968. 75. Tanaka, K. R., Valentine, W. N., and Miwa, S.: Pyruvate kinase (PK) deficiency hereditary nonspherocytic hemolytiC anemia. Blood, 18:784, 1961; 19:267, 1962. 76. Tanaka, K. R., Valentine, W. N., and Miwa, S.: Studies on hereditary spherocytosis and other hemolytiC anemias. Clin. Res., 10:109,1962. 77. Tarui, S., Kono, N., Nasu, T., and Nishikawa, M.: Enzymatic basis for the coexistence of myopathy and hemolytic disease in inherited muscle phosphofructo-kinase deficiency. Biochem. Biophys. Res. Commun., 34:77,1969. 78. Valentine, W. N.: Deficiencies associated with Embden-Meyerhof pathway and other metabolic pathways. Sem. Hemat., 8:348, 1971. 79. Valentine, W. N.: Hereditary hemolytic anemias associated with specific erythrocyte enzymopathies. Calif. Med., 108:280,1968. 80. Valentine, W. N., Anderson, H. M., Paglia, D. E., et aL: Nonspherocytic-hemolvtic ane· mia, high red cell ATP, and ribose-phosphate pyrophospho-kinase (RPK E.C. 2.7.6.1) deficiency. Clin. Res., 19:567, 1971. 81. Valentine, W. N., Asieh, H. S., Paglia, D. E., et al.: Hereditary hemolytic anemia associated with phosphoglycerate kinase deficiency in erythrocytes and leukocytes. A probable x-chromosome-linked syndrome. New Eng. J. Med., 280:528, 1969. 82. Valentine, W. N., Oski, F. A., Paglia, D. E., et al.: Hereditary hemolytic anemia with hexokinase deficiency. Role of hexokinase in erythrocyte aging. New Eng. J. Med., 276:1,1967. 83. Valentine, W. N., and Tanaka, K. R.: Pyruvate kinase and other enzyme deficiency hereditary hemolytic anemias. In Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds.: The Metabolic Basis oflnherited Disease. New York, McGraw-Hill, 2nd ed., 1966. 84. Van Slyck, E. J., and Rebuck, J. W.: Elliptocytosis and sickle cell trait. Arch. Intern. Med., 114:657, 1964.

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