ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 12, No. 3Copyright © 1982, Institute for Clinical Science, Inc.

Red Cell Enzymopathies in the Newborn II. Inherited Deficiencies of Red Cell Enzymes*SUSAN F. TRAVIS, M.D.

Departm ent o f Pediatrics and Cardeza Foundation fo r Hematologic Research,

Thomas Jefferson University, Philadelphia, PA 19107

ABSTRACTThe mature, anueleate human red cell is incapable of synthesizing en­

zymes and is dependent upon anaerobic metabolism to m eet its energy requirements. Thus, inherited red cell enzyme deficiencies of the glycolytic pathway often result in chronic nonspherocytic hemolytic anemia (CNSHA). The hexosemonophosphate (HMP) shunt is normally relatively inactive and enzyme deficiences, such as G-6-PD deficiency, usually result in episodic hemolysis secondary to oxidative stress. Deficiency of G-6-PD is the most common red cell enzymopathy. Pyruvate kinase (PK) deficiency is the most common enzyme deficiency of the glycolytic pathway, followed in fre­quency by glucose phosphate isomerase (GPI) deficiency. Other glycolytic enzyme deficiencies are rare. Red cell phosphofructokinase deficiency can be associated with muscle weakness; triosephosphate isomerase (TPI) and phosphoglycerate kinase deficiencies with neurologic abnormalities. Since its original description, pyrimidine-5'-nucleotidase deficiency has been rec­ognized with greater frequency as a cause of CNSHA. Enolase, glyceralde- hyde phosphate dehydrogenase, and lactic dehydrogenase deficiencies are not associated with hemolysis.

The newborn with CNSHA can develop anemia and significant jaundice in the first 24 hrs of life. Precise diagnosis may be difficult owing to the volume of blood required and the laboratory facilities available. Screening tests for G-6-PD, GPI, TPI, and PK deficiencies and the red cell concentra­tion of 2,3-diphosphoglycerate can all be done with small amounts of blood obtained with a heel puncture.

Data obtained for red cell glycolytic intermediates and quantitative en­zyme assays must be compared to normal values for neonates since red cells at birth and during the first two months of life have unique metabolic characteristics.

* Supported in part by NIH Grant HD-10213.163

0091-7370/82/0500-0163 $02.00 © Institute for Clinical Science, Inc.

164 T R A V IS

IntroductionThe mature, anucleate, human red cell

is devoid of mitochondria and other in­tracellular organelles and is incapable of synthesizing hemoglobin, membrane pro­tein or metabolic enzymes. It lacks an in­tact citric acid cycle and is essentially dependent upon anaerobic metabolism to m eet its energy requirem ents. Under nor­mal circumstances, both the metabolic ca­pacity and energy demands are delicately balanced so that the red cell survives 120 days and can fulfill its primary role, which is to deliver oxygen from the lungs to the tissues.

An inherited enzyme deficiency that results in disruption of this delicate bal­ance could result in premature destruc­tion of the red cell and, by definition, hemolytic anemia. The purpose of this pa­per is to review the metabolic and clini­cal aspects of these enzyme deficiencies, with particular reference to the newborn period. More comprehensive reviews of red cell enzymopathies have been pub­lished recently.47,48,64,88

Red Cell MetabolismThe mature human red cell is basically

an anaerobic cell. Glucose is the normal substrate which enters the red cell by a process of facilitated diffusion93; the intra­cellular concentration approximates, on a water basis, that in the extracellular envi­ronment. Within the red cell, approxi­mately 97 percent is phosphorylated to glucose-6-phosphate (G-6-P) via the hexo- kinase (HK) reaction. Metabolism of G-6-P then proceeds through the hexose mono­phosphate (HMP) shunt or the Embden- M eyerhof (glycolytic) pathway.

Under normal conditions, 90 percent or more of the G-6-P is metabolized via the glycolytic pathway, with the conversion of one mole of G-6-P to two moles of lactic acid. Metabolism of one mole of glucose via this pathway results in the net produc­tion of two moles of adenosine triphos­phate (ATP). The ATP serves as an energy source for maintenance of the normal bi­

concave disc shape of the red cell and plasticity of the red cell membrane, mem­brane lipid, active cationic pumping of potassium into the cell and sodium out of the cell, prevention of calcium accumula­tion in the red cell membrane, and gluta­thione synthesis.

The glycolytic pathway also provides reduced pyridine nucleotide (NADH) at the glyceraldehyde-3-phosphate dehydro­genase step, which can be utilized by the NADH-dependent methemoglobin re­ductase to reduce methemoglobin formed within the red cell. The Rapoport-Lue- bering or 2,3-diphosphoglycerate (2,3- DPG) shunt is an important alternate met­abolic pathway of glycolysis, whereby1.3-diphosphoglycerate is converted to2.3-DPG by the enzyme, 2,3-diphospho­glycerate mutase (DPGM). The 2,3-DPG combines with deoxyhemoglobin and re­duces the affinity of hemoglobin for oxy­gen,6,18 thereby shifting the oxygen-he- moglobin equilibrium curve to the right, facilitating the unloading of oxygen from the red cell to the tissue.

Metabolism of glucose via the HMP shunt results in the net production of two moles of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and one mole of C 0 2 for every mole of G-6-P me­tabolized. The NADPH generated via this pathway is vital to the red cell since it acts as a co-factor to maintain glutathione in the reduced state (GSH) via the gluta­thione reductase reaction. Thus, GSH, in turn, protects sulfhydryl groups of hemo­globin against oxidative dénaturation, as well as stabilizing certain intracellular en­zymes. GSH detoxifies the primary dé­naturant, hydrogen peroxide, by serving as a substrate for the enzyme glutathione peroxidase.

Thus, glucose metabolism via the gly­colytic pathway: (1) fulfills the energy re­quirements of the red cell by providing ATP which maintains the integrity and deformability of the red cell membrane, (2) provides 2,3-DPG via the Rapoport- Luebering shunt which enables the red cell to modulate its hemoglobin-oxygen

RED CELL ENZYM OPATHIES 165affinity, and (3) provides NADH that can be utilized by the NADH-dependent met- hemoglobin reductase.

Metabolism via the HMP shunt pro­vides a means of protecting the red cell against oxidative damage. Perturbations of metabolism caused by inherited de­fects of the enzymes of either of these pathways can disrupt the harmonious bal­ance of structure and function usually present in the mature red cell and lead to premature cell death.General Clinical and Laboratory Features

The congenital non-spherocytic hemo­lytic anemias (CNSHA) share many clini­cal characteristics. These include: (1) life­long hemolysis; (2) hyperbilirubinemia, usually with a history of neonatal jaun­dice, which may have required exchange transfusion; (3) a variable degree of sple­nomegaly, which tends to increase with age; (4) an increased incidence of chole­lithiasis; (5) increased severity of anemia with infection; (6) variable severity of anemia, ranging from a mild compensated state to a severe hemolytic process which is transfusion dependent; (7) mild to mod­erate improvement after splenectomy, but not cure of the hemolytic process as ob­served in hereditary spherocytosis; and (8) familial incidence.

Deficiencies involving enzymes of the glycolytic pathway result in CNSHA in contrast to defects in the HMP shunt which usually result in acute episodic hemolysis secondary to oxidative stress, infection, or acidosis, although there are variants of G-6-PD with CNSHA.

The laboratory findings in general are variable, and precise diagnosis cannot be made without specific tests. The red cell morphology reflects the hemolytic state and young red cell population and in­cludes a variable degree of macro- and anisocytosis and polychromasia. Spicu- lated microspherocytes are present in glu- cosephosphate isomerase deficiency and irregularly contracted cells are observed

in pyruvate kinase deficiency and other enzyme deficiencies of the glycolytic path­way. In G-6-PD deficiency, during a he­molytic episode, occasional spherocytes, red cell fragmentation, and Heinz bodies may be observed. Basophilic stippling is prom inent in pyrimidine-5'-nucleotidase deficiency.83

Other non-specific laboratory features include hyperbilirubinem ia of the indi­rect type, low plasma haptoglobin and hemopexin, and shortened autologous51 Cr red cell survival. The osmotic fragility is usually normal and may be increased or decreased at 24 hours, unlike hereditary spherocytosis which results in a uniform increase in osmotic fragility after incuba­tion. The autohemolysis test serves as a non-specific screening test and is rarely used.

A useful adjunct in the diagnosis of glycolytic enzyme deficiencies is analysis of the pattern of glycolytic interm edi­ates and adenosine triphosphate (ATP). A block in glycolysis at a specific enzyme step should result in an increase in the concentration of substrates prior to that step and a reciprocal decrease in those distal to the step, and the crossover site may then offer a rational approach as to the specific enzyme to be tested. Simi­larly, a decreased concentration of GSH may reflect an enzyme deficiency in the HMP shunt or glutathione metabolism.

The most common enzymopathy is G-6- PD deficiency.11 Deficiency of PK is the most common enzyme defect in the gly­colytic pathway, followed by GPI defici­ency; the other deficiencies, such as HK, PFK and DPGM, are rare; Deficiency of PD-5'-N has been diagnosed with greater frequency since its description.11

Hereditary Enzyme Deficiencies of the Glycolytic PathwayH e x o k in a s e D e f i c i e n c y

Hexokinase (HK) catalyzes the phos­phorylation of glucose to glucose-6-phos-

166 TRAVIS

phate (G-6-P):G lucose + A TP G lucose-6-P + A D P

This is the initial enzymatic step that allows glucose to enter the glycolytic or HMP pathways. The activity of HK in the human red cell is normally very low, and it is one of the rate limiting steps that con­trols glycolysis. Deficiency of HK gener­ally results in a decreased rate of red cell glucose consumption, decreased concen­trations of G-6-P, and other intermediates distal to this step and reduced red cell ATP. The glutathione content and glu­tathione stability are normal.

Deficiency of HK results in a chronic hemolytic state and has been associated with hyperbilirubinem ia in the newborn. It has also been described in three cases of Fanconi’s aplastic anemia,41 but it is not characteristic of this disorder. Splenec­tomy usually results in improvement re­garding transfusion requirements, but an uncompensated hemolytic state persists.

Although this disorder is rare, genetic polymorphism is apparent. An autosomal recessive mode of inheritance has been proposed, but Necheles et a l54 have re­ported a family where both the father and son were affected. W hen red cell HK is studied electrophoretically, four bands have been described,2 a predominant tri­ple HK, Type I, and a more anodal faint HK, Type III.66 Deficient kindreds of HK have been reported that have: (1) a nor­mal electrophoretic pattern but the en­zyme is unstable in hemolysates, and has an altered substrate affinity34; (2) absence of all bands except band 1 with normal enzyme kinetics2; (3) absence of Type III with altered substrate affinity54; and (4) others.47,66

Activity of HK is markedly increased in reticulocytes and its activity declines markedly as the red cell ages.85 Thus, it should be emphasized that the HK activ­ity in the red cells of a suspected deficient patient must be compared to that in cells of a similar mean age. By employing retic­ulocyte rich blood as a control, it becomes

readily apparent that the “normal” activ­ity that may be obtained in some HK defi­cient subjects with marked reticulocytosis is, in reality, greatly diminished for red cell age. This is particularly pertinent in evaluating the newborn with hemolysis, since HK in cord blood is normally very elevated.

The concentration of G-6-P is also in­creased in young red cells, so that “nor­mal” G-6-P may be obtained in some HK deficient subjects rather than the ex­pected decrease in this glycolytic inter­mediate. This is particularly relevant to the newborn since the concentration of G-6-P in normal infants is elevated, out of proportion to the age of the red cell popu­lation until one to two months of age and then declines to levels that are appropri­ately increased for mean cell age.80

The concentration of 2,3-DPG is de­creased in HK deficiency, and the sub­ject cannot compensate for his anemia with an increase in 2,3-DPG resulting in decreased exercise tolerance for the de­gree of anemia.20 This decrease in 2,3- DPG has little clinical application in the newborn owing to the presence of fetal hemoglobin.

G l u c o s e p h o s p h a t e I s o m e r a s e D e f i c i e n c y

Glucosephosphate isomerase (GPI) cat­alyzes the reaction:

Glucose-6-P <--------> Fructose-6-PDeficiency of red cell GPI results in a

moderate chronic hemolytic process.5,58 Isoenzymes of GPI from different tissues have the same electrophoretic properties, and decreased activity has been demon­strated in leukocytes, skin fibroblasts, and platelets in GPI deficient subjects. There is no associated leukocyte dysfunction or systemic disease except one report of a subject with mental retardation and in­creased hepatic glycogen.89 Deficiency of GPI is clearly inherited as an autosomal recessive trait; heterozygotes have ap­proximately one-half normal activity and

R E D C E L L E N Z Y M O P A T H IE S 167no evidence of hemolysis. Electropho­retic studies have revealed the genetic polymorphism of this disorder.

Splenectomy results in moderate im­provement, but an uncompensated hemo­lytic state persists. Attempts to bypass the block in glycolysis by stimulating metabo­lism of G-6-P through the HMP shunt with such agents as ascorbic acid5 or methylene blue have been unsuccessful in improving the anemia in GPI deficiency.

Neonatal jaundice is not uncommon58 and may require exchange transfusion. Al­though specific morphologic findings are uncommon in glycolytic enzyme defi­ciencies, spherocytes have been reported in this disorder.56

Deficiency of GPI results in an in­creased concentration of red cell G-6-P and decreased concentration of fructose- 6-phosphate (F-6-P). Since the concentra­tions of G-6-P and F-6-P are normally ele­vated in red cells with a young mean cell age and are markedly increased in the first month of postnatal life,80 a low F-6-P in a newborn with hemolysis is of particular significance and highly suggestive of GPI deficiency. A reliable screening test for detecting GPI deficiency is available and is particularly helpful in the neonate since it can be done by obtaining blood from a heel puncture.15

P h o s p h o f r u c t o k i n a s e D e f i c i e n c y

Phosphofructokinase (PFK) catalyzes the reaction:

Fructose-6-P + ATP < —»Fructose-1,6-diphosphate + ATP

PFK is one of the major rate limit­ing enzymes in the control of red cell glycolysis. Deficiency of this enzyme has been associated with a severe myopathy referred to as Tarui’s disease or Type VII glycogen storage disease. This disorder is characterized by virtual absence of skel­etal muscle PFK and approximately 50 percent of normal red cell PFK activ­ity.39,78 These patients have a mild

non-spherocytic hemolytic anemia. Auto­somal recessive inheritance has been postulated.

Deficiency of PFK without myopathy has also been described. In one of these individuals, hemolytic anemia and sple­nomegaly was associated with a 50 per­cent reduction in red cell enzyme activity, and an unstable enzyme with altered ki­netic properties.92 In another family w ith­out muscle involvement, red cell PFK ac­tivity was barely detectable.50 Vora et a l91 have defined the molecular basis of PFK deficiency by demonstrating that PFK is composed of two separate subunits, M (muscle-type) and L (liver-type) and red cell PFK is a mixture of five tetrameric isozymes: M4, M3L, M2L2, ML3, andL 4. In type VII glycogenolysis, the M subunit was absent, so that red cell PFK consisted exclusively of the L4 isozyme.90

Hemolysis is generally mild in this dis­order. Deficiency of PFK results in an in­creased concentration of red cell G-6-P and F-6-P and a decreased concentration of2,3-DPG. Subjects with PFK deficiency may not be anemic owing to compensa­tory erythrocytosis secondary to this de­crease in 2,3-DPG.90

Red cell G-6-P and F-6-P are increased and PFK activity is decreased in normal infants during the first two months of life.80 In neonates with a hemolytic proc­ess, these transiently abnormal metabolic characteristics should not be mistaken for congenital PFK deficiency.

A l d o l a s e D e f i c i e n c y

Aldolase (ALD) catalyzes the reaction:Fructose-1,6-diphosphate «--------- »

glyceraldehyde-3-phosphate +dihydroxyacetone phosphate

A single patient with aldolase defi­ciency has been reported.13 The parents were first cousins. Deficiency of ALD was associated with moderate mental retardation, increased hepatic glycogen, and mild CNSHA.

168 TRAVIS

T r i o s e p h o s p h a t e I s o m e r a s e D e f i c i e n c y

Triosephosphate isomerase (TPI) cata­lyzes the reaction:Dihydroxyacetone phosphate <-------- >

Glyceraldehyde-3-phosphateRed cell TPI deficiency is associated

with a moderate to marked hemolytic ane­mia. In all cases reported to date, a severe and usually progressive neurologic disor­der is present that begins after 6 months of age.3.7° Thg enzyme deficiency is trans­mitted as an autosomal recessive trait and is widespread, involving leukocytes, spi­nal fluid, muscle, and skin fibroblasts. An increased susceptibility to infections has been reported. Three affected indi­viduals have died suddenly from pre­sumed cardiac arrest, which is suggestive of myocardial involvement in this disor­der.3,70 It is postulated that the general­ized nature of the enzyme deficiency is responsible for the spectrum of clinical symptoms present in this disorder. Het­erozygotes have approximately one-half normal red cell TPI activity and are clini­cally asymptomatic.

Two instances of cri-du-chat syn­drom e74 in association with decreased red cell TPI activity have been reported. Other cases, however, have not been asso­ciated with decreased TPI activity.

Deficiency of TPI results in marked accumulation of dihydroxyacetone phos­phate.

Although the systemic manifestations of this disorder develop early, they are generally not present in the newborn pe­riod. Hemolytic anemia with jaundice, however, does present in the neonate. A screening test is available that can be per­formed with blood obtained from a heel stick.33

2 ,3 -D i p h o s p h o g l y c e r a t e M u t a s e D e f i c i e n c y

2,3-Diphosphoglycerate mutase (2,3- DPGM) catalyzes the reaction:

3-PG1,3-Diphosphoglycerate-------->•2,3-diphosphoglycerate

This reaction represents the proximal limb of the Rapoport-Luebering shunt. 3-Phosphoglycerate (3-PG) is a necessary co-factor. This shunt represents the only point in the red cell where net synthesis of2,3-DPG occurs. 2,3-DPG is converted to 3-PG by the enzyme, diphosphoglycerate phosphatase (DPGP). It appears that DPGM, DPGP, and monophosphoglyc- eromutase represent active sites on the same molecule.67,69

Red cell DPGM deficiency with chronic hemolytic anemia associated with mild jaundice has been reported in sev­eral patients.1,40,71,81 In two of these case reports, much of the evidence was indi­rect,1,40 based on a decreased content of red cell 2,3-DPG. In another family,71 the parents of a child with a severe hemolytic process had 50 percent of normal red cell DPGM activity, but the enzyme could not be m easured in the child owing to the presence of large amounts of transfused blood. It was presumed that the parents, who were clinically unaffected were het­erozygotes and the infant was homozy­gous, suggesting an autosomal recessive mode of inheritance. Autosomal dominant inheritance was demonstrated in three generations of a kindred with DPGM de­ficiency and well compensated hemol­ysis.81 Activity of DPGM was reduced to52 to 61 percent of normal in affected members, DPGP was reduced to 40 per­cent of normal, and MPGM was normal. The hemoglobin was normal but hemol­ysis was documented with elevated retic­ulocyte counts, decreased haptoglobins, and decreased 51Cr red cell survival. No other cause for hemolysis was identified. In contrast, a patient with virtually com­plete absence of DPGM and 2,3-DPG was healthy and had no evidence of hemol­ysis.68 His only manifestation was poly­cythemia. There is obviously marked vari­ation in the clinical expression of this disorder.

R E D C E L L E N Z Y M O P A T H IE S 169Deficiency of DPGM is not known to be

associated with problems in the newborn period.P h o s p h o g l y c e r a t e K i n a s e

D e f i c i e n c y

Phosphoglycerate kinase (PGK) cata­lyzes the reaction:

Mg++1.3-Diphosphoglycerate + ADP <------- »3-Phosphoglycerate 4- ATP

Red cell PGK deficiency has been stud­ied in a large Chinese kindred.82 The in­heritance of this disorder is X-linked. Af­fected males had a moderately severe hemolytic anemia, with marked defici­ency of erythrocyte and leukocyte PGK and mild mental retardation with behav­ioral disturbances. Increased susceptibil­ity to infection has not been described.

Other cases of PGK deficiency4,36 have been reported and are similar. However, one PGK variant, PGK Miinchen, had moderately decreased enzyme activity, thermal instability, and no clinical symp­toms.38 Recently, this variant was shown to be secondary to one amino acid substi­tution, an asparagine for an aspartic acid.26

In PGK deficiency, glycolysis is “blocked” at an ATP generating step, but1.3-diphosphoglycerate can be metabo­lized to lactate via the Rapoport-Lueber- ing shunt. This “bypass” results in no net gain of ATP. There is marked elevation of the red cell 2,3-DPG concentration. Hem­olytic anemia and jaundice can appear in the newborn period,36 bu t the neuro­logic signs generally do not appear until after the first year of life.P y r u v a t e K i n a s e D e f i c i e n c y

Pyruvate kinase (PK) catalyzes the reaction:Phosphoenolpyruvate + ADP < ^g++—

pyruvate + ATPDeficiency of PK is the most common

and best described enzyme deficiency of

the glycolytic pathway, and recent com­prehensive reviews are available.47,88 Its distribution is world wide but is most prevalent in persons of Northern Euro­pean extraction. In the United States, a particularly high incidence has been noted among the Amish in Mifflin County, Pennsylvania.16

Deficiency of PK is inherited as an autosomal recessive trait. The heterozy­gotes have approximately 50 percent of normal PK activity in their red cells and are unaffected clinically. The homozy­gous deficient individuals demonstrate variability in the clinical expression of their disease, ranging from a mild com­pensated hemolytic process to a severe, transfusion-dependent hemolytic state that may be life-threatening. This broad clini­cal spectrum may be present in the same family. The PK activity of leuko­cytes is normal. Acquired PK deficiency has been described in hematologic disor­ders, such as malignant and dyserythro­poietic states.31

The majority of patients exhibit m oder­ate to severe anemia, hyperbilirubinem ia, and splenomegaly. Cholelithiasis at an early age is not uncommon. Most in­stances of PK deficiency are noted in in­fancy or childhood, but rare cases escape detection until adult life. Anemia and hyperbilirubinem ia, requiring exchange transfusion, have been observed in new­borns, often in the first 24 hours of life, and kernicterus has been reported.57 In severely affected children, w idened di­ploic spaces and a “hair on end” appear­ance of the skull can be seen radiograph- ically, similar to the roentgenographic changes observed in thalassemia major.17 Infection is usually associated with an ac­celerated rate of hemolysis and/or tran­sient hypoplastic crisis.

Splenectomy usually affords some de­gree of improvement with a decrease in or elimination of transfusion requirem ents and variable rise in hemoglobin level, but an uncompensated hemolytic state per­sists. The reticulocyte count increases

170 T R A V IS

markedly post-splenectomy, sometimes to levels as high as 80 percent. It has been demonstrated that PK-deficient reticulo­cytes are sequestered in the spleen in pa­tients with this disorder.52 It has been pro­posed that these metabolically affected young cells are doomed to early ex­tinction, but splenectomy allows them to survive longer in the circulation and ac­counts for the marked increase in reticulo­cyte counts observed after removal of the spleen.

The peripheral smear in PK deficiency is not specific and is characterized by macrocytosis, polychromasia, and a vari­able num ber of irregularly contracted red cells. These abnormal appearing red cells increase in num ber after splenectomy. A variable num ber of normoblasts may be present. The autohemolysis pattern that had previously been considered charac­teristic of PK deficiency is Dacie type I I 72 (marked hemolysis in saline and glucose, with correction with ATP). This pattern is not only variable but is non-specific.

Genetic polymorphism is common in PK deficiency. Variants with altered sub­strate affinity for phosphoenolpyruvate (PEP)30,55 and abnormalities in fructose diphosphate activation,59 ATP inhibition and thermal stability73 are frequently noted. “ Homozygous” PK deficient sub­jects that are the product of non-consan- guineous matings are usually doubly het­erozygous for two different red cell PK variants.32 Those with increased substrate requirem ents (high KroPEP) usually have normal or slightly decreased enzyme ac­tivity,60 since optimal concentrations of PEP are employed in the PK assay system and this concentration is far in excess of that found in red cells. Since the dis­covery of these mutants, PK activity is routinely measured at high and low PEP concentrations. A screening test for PK deficiency is available7 and useful, but a negative screen does not rule out PK defi­ciency owing to these kinetic mutants with normal activity.

Like HK, PK is an age dependent enzyme. Leukocyte PK activity is many

times greater than red cell activity and is a different isozyme. Thus, the white cells of PK deficient subjects have normal activ­ity, and it is important to remove the white cells prior to PK assay.

Red cells deficient in PK are charac­terized by impaired glycolysis and in­creased concentrations of 2,3-DPG and PEP. Reduced concentrations of red cell ATP would be anticipated owing to a block in glycolysis at an ATP generating step. M easurement of red cell ATP has yielded variable results, however, since the patients who have extreme reticulo- cytosis are capable of maintaining red cell ATP via oxidative phosphorylation.O t h e r G l y c o l y t i c E n z y m e D e f i c i e n c i e s

Red cell enolase deficiency75 has been reported in two sisters who exhibited a mild CNSHA and an accelerated rate of hemolysis after ingestion of nitrofuran­toin. No other case of enolase deficiency has been described. Red cell glycer- aldehyde phosphate dehydrogenase and lactic dehydrogenase49 deficiencies have been reported but were unassociated with hemolytic anemia.Deficiencies of the Hexose Monophosphate ShuntG l u c o s e -6 -P h o s p h a t e D e h y d r o g e n a s e (G -6 -P D ) D e f i c i e n c y

Deficiency of G-6-PD43,61 is the most common inborn error of metabolism, and extensive investigations have made this abnormality the best understood and most thoroughly studied red cell enzymopathy. It is widely distributed geographically and occurs with greatest frequency among Blacks, Chinese, and Caucasians of Med­iterranean ancestry, such as Italians, Greeks, and Sephardic Jews. The geo­graphic distribution of G-6-PD deficiency is strikingly similar to that of the epi- demicity of falciparum malaria. Luzzatto et a l44 demonstrated that when the red cells of heterozygous G-6-PD deficient fe­

R E D C E L L E N Z Y M O P A T H IE S 171males were simultaneously stained for both G-6-PD activity and the malarial parasite, the normal red cells were more heavily parasitized than the deficient cells and it is likely that the high fre­quency of G-6-PD deficiency in malarial zones was maintained by the advantage conferred upon the heterozygous female.

The incidence of G-6-PD deficiency varies widely in different parts of the world. For example, in the United States approximately 11 percent of Black males63 and 1 percent of Black females are G-6-PD deficient, whereas in certain African tribes the incidence is as high as 28 per­cent among males. Italian Americans have an incidence of G-6-PD deficiency of ap­proximately 2 percent; in Sardinia, it is as high as 30 percent.35,46

The X-linked nature of this disorder is well established.19 Owing to the random inactivation of one of the two X chromo­somes,45 the heterozygous female (X+X_) can have low, intermediate, or normal G- 6-PD activity.

Over 160 variants of G-6-PD have been described. These enzymes are classified9 into five major groups based on enzyme activity:

I. Severe enzyme deficiency associated with chronic nonspherocytic hemolytic anemia; II. Severe enzyme deficiency;III. Moderate to mild enzyme deficiency;IV. Very mild or no enzyme deficiency; and V. Increased enzyme activity. They are further classified according to electro­phoretic mobility (fast, normal, or slow). Further characterization is made within each group utilizing different biochemi­cal techniques, such as the Michaelis con­stant (Km), pH activity curves; heat dena- turation; and capacity of the enzyme to utilize substrate analogues.

The normal enzyme (or wild type) is designated as B and is the most prevalent type. An electrophoretically fast variant with slightly reduced activity that is un­associated with hemolysis is found in approximately 20 percent of American Blacks and is designated as type A. The most common deficient variants are G-6-

PD A-, found in Blacks, G-6-PD M editer­ranean, prevalent in Italians, Greeks, and Sephardic Jews; and possibly G-6-PD Canton and Hong Kong-Pokfulam com­mon in Orientals. Fingerprinting tech­niques have demonstrated that the differ­ence between the mutant type A and the wild type B resides in one amino acid substitution, an aspartic acid for an asparagine, respectively.95

Current observations suggest that the deficiency in the common variants, G-6- PD A- and G-6-PD Mediterranean is due to an unstable enzyme.62 The normal en­zyme has a half-life (t lh) of 62 days. In comparison, G-6-PD A- has a t l/i of 13 days. The instability of G-6-PD M editer­ranean is so great that activity can only be detected in reticulocytes. Since G-6-PD is an age-dependent enzyme, the differ­ences in the life-span of the enzyme have clinical relevance. In the G-6-PD A-, in­gestion of an “oxidant” drug results in de­struction of old G-6-PD deficient red cells and compensatory hyperactive erythro- poiesis producing a young red cell popu­lation with near-normal G-6-PD activity, which is resistant to further “oxidant” stress. Thus, the hemolytic process is self- limited, despite further ingestion of the drug. The G-6-PD Mediterranean enzyme, however, is inactivated so rapidly that reticulocytes emerging from the bone marrow already have decreased G-6-PD activity; the increase in G-6-PD activity during a hemolytic episode is barely de­tectable, and hemolysis may not be self­limited.

G-6-PD is the first enzyme of the HMP shunt and catalyzes the conversion of G-6- P to 6-phosphogluconate. 6-Phosphoglu- conate is further metabolized to ribulose-5-P and C 0 2. The ribulose-5-P is then converted to F-6-P or G-3-P through the action of transketolase or transaldolase. These substrates then re-enter the gly­colytic pathway. The F-6-P can be metabo­lized to lactic acid or to glucose-6-P and then be recycled through the HMP shunt.

For every mole of G-6-P that enters the HMP shunt, two inoles of NADPH are

1 7 2 T R A V IS

produced. In turn, NADPH serves to main­tain glutathione in the reduced state (GSH) by serving as a co-factor for the enzyme glutathione reductase (GR). The GSH reduces hydrogen peroxide (H20 2) to H20 in the glutathione peroxidase (GSH-Px) reaction. NADPH also partici­pates as a coenzyme in the minor pathway of methemoglobin reduction.

Under normal circumstances, the HMP shunt is relatively inactive, and metabo­lism proceeds via the glycolytic pathway. Activity of G-6-PD is stimulated by in­creased concentrations of erythrocyte NADP.76 “Oxidant” compounds appear to decrease the concentration of NADPH and, thereby, increase the concentration of NADP. Through these mechanisms, G-6-PD can counterbalance “oxidant stress” by accelerating flow through the HMP shunt. Subjects deficient in G-6-PD are incapable of compensating in this manner and little NADPH regeneration can occur. In the absence of an intact NADPH gener­ating system, mixed disulfides accumu­late in the cell, which leads to instability and heme loss with irreversible denatur- ation and precipitation of globin as Heinz bodies.

Persons deficient in G-6-PD are clin­ically asymptomatic unless challenged by “oxidant stress,” which may be secondary to a drug or its active metabolite, infec­tion, especially hepatitis, and acidosis.

A certain num ber of G-6-PD variants manifest a moderate to severe chronic he­molytic state (CNSHA)43,65 in addition to acute exacerbations secondary to drug ad­ministration and/or infection. The exact mechanism for chronic hemolysis in these patients is not completely understood.

Deficiency of G-6-PD is often impli­cated as a cause of neonatal jaundice and kernicterus in otherwise healthy new­borns. Term American Black infants94 do not have a significantly increased inci­dence of jaundice, but term Jamaican,27 African14 and prem ature24 Black neonates and both term and premature Cauca­sian23,25 and O riental42 infants are at in­

creased risk. The appearance of jaundice in otherwise healthy neonates with G-6- PD deficiency tends to be familial.25

The peak bilirubin is usually attained at three to four days and may remain ele­vated for one to two weeks. The hyper­bilirubinem ia may be severe and require exchange transfusion to prevent kernic­terus. Variants with CNSHA tend to de­velop jaundice in the first 24 hours and may require early exchange transfusion.65

In an infant with G-6-PD deficiency and hemolysis, a search for an initiating mechanism should be sought, such as ma­ternal drug ingestion, infection, hypoxia, or acidosis. Other causes of hemolysis should be considered, since G-6-PD de­ficiency may be an “innocent bystander” in certain unresolved cases of neonatal jaundice.

Parents of G-6-PD deficient infants should be advised concerning avoidance of certain drugs and naphthalene contain­ing mothballs. Mothers who are nursing G-6-PD deficient infants should take sim­ilar precautions, since these agents may be excreted into breast milk. Heterozy­gous females should also be advised against the use of any of these offending agents in future pregnancies, since they may cross the placenta.

The diagnosis of G-6-PD deficiency can be made with relative ease employing a screening test10 or the quantitative spec- trophotometric enzyme assay.10 It should be reemphasized that the Black deficient with reticulocytosis secondary to drug in­duced hemolysis or other causes, such as sickle cell anemia, may have a normal G-6-PD screening test. When reticulocytosis is present in G-6-PD Mediterranean, the screening test can be used with confi­dence. Heterozygous females are more difficult to diagnose. Comparison of the enzyme activity in subjects with G-6-PD A- and reticulocytosis with non-deficient red cells of a similar mean age will reveal that the “low-normal” activity is, in re­ality, greatly diminished for red cell age.

R E D C E L L E N Z Y M O P A T H IE S 1736 -P h o s p h o g l u c o n a t e D e h y d r o g e n a s e (6 -P G D ) D e f i c i e n c y

The conversion of 6-phosphogluconate to ribulose-5-P and C 0 2 is catalyzed by6-PGD. Deficiency of this enzym e21 is ex­tremely rare, and there is usually no evi­dence of hemolysis.

G l u t a t h i o n e R e d u c t a s e (G R ) D e f i c i e n c y

Glutathione reductase, as discussed pre­viously, catalyzes the reduction of oxi­dized glutathione (GSSG) to reduced glu­tathione (GSH) with NADPH serving as the hydrogen donor. GR exists in two forms, the active flavin-adenine dinucleo­tide (FAD)-bound form and an inactive form, lacking FAD. Reduction of red cell GR to half normal activity does not impair the rate of reduction of GSSG to GSH. Partial deficiency of GR is quite common and has been reported as an inherited de­ficiency and found in many diverse disor­ders, including pancytopenia and hemo­globin C disease. It has recently been recognized that the majority of cases with GR deficiency were dietary in origin and secondary to riboflavin deficiency; ad­ministration of riboflavin to these individ­uals restored GR activity to normal.8 The relationship of GR deficiency to hemato­logic disease is, at present, ill-defined and of questionable significance.11G l u t a t h i o n e P e r o x id a s e D e f i c i e n c y

Glutathione peroxidase (GSH-Px) cata­lyzes the conversion of H20 2 to H20 . D e­ficiency of this enzym e53 is transmitted as an autosomal recessive trait and has been reported to be associated with CNSHA, Heinz body formation, drug-induced ac­celeration of hemolysis, and neonatal jaundice.

However, transient GSH-Px defici­ency28 is a normal finding in neonates and has not been associated with neonatal hyperbilirubinemia. GSH-Px is a sele­nium containing enzyme, and acquired

GSH-Px deficiency79 is associated with selenium deficiency; there is no evidence of hemolysis in these subjects. Also, 30 percent of individuals of Jewish ancestry were found to have GSH-Px activity that was half normal and had no evidence of hemolysis.12 Thus, it is probable that GSH-Px deficiency is not a cause of CNSHA11 or neonatal jaundice.G l u t a t h i o n e D e f i c i e n c y

Synthesis of GSH occurs in two steps. In the first step, glutamic acid and cys­teine form glutamyl-cysteine; in the sec­ond step, glycine combines with gluta­myl-cysteine to form the tripeptide, GSH. In affected individuals, either the first step (gammaglutamyl-cysteine synthetase deficiency)51 or the second step (glu­tathione synthetase deficiency)37 can be involved.

Deficiency of GSH is transmitted as an autosomal recessive trait. Affected in­dividuals manifest a mild CNSHA and drug-induced exacerbation of hemolysis. Generalized glutathione synthetase defi­ciency has been associated with oxopro- linuria, chronic metabolic acidosis and, in some instances, neurologic findings.29Disorders of Nucleotide MetabolismP y r i m i d i n e -5 ' -N u c l e o t i d a s e D e f i c i e n c y

Pyrimidine-5'-nucleotidase (PD-5'-N) catalyzes the reaction:UridineCytidine Monophosphate + H20 —> Thymidine

UridineCytidine + Inorganic phosphate Thymidine

Deficiency of PD-5'-N is associated84,87 with mild to moderate CNSHA. Neona­tal hyperbilirubinem ia has been reported. Markedly increased levels of red cell “ATP” and total “adenine nucleotides,” elevated glutathione levels, and promi­

174 T R A V IS

nent basophilic stippling of the red cells are characteristic. The increased concen­tration of ATP reported originally is spu­rious since they are pyrimidine nucleo­tides. Deficiency of PD-5'-N also occurs as an acquired disease as a result of lead poisoning.86

A d e n y l a t e K i n a s e D e f i c i e n c y

Adenylate kinase (AK) catalyzes the reaction:

2 ADP «--------> ATP + AMPDeficiency of AK77 is associated with a

moderate CNSHA and is probably inher­ited as an autosomal recessive trait. The parents of the affected subjects have inter­mediate enzyme activity and no hemato­logic abnormality. The mechanism of the hemolysis has not been defined.I n c r e a s e d A d e n o s i n e D e a m in a s e A c t i v i t y

Adenosine deaminase (ADA) catalyzes the reaction:Adenosine + H20 -------->

Inosine + NH3Decreased ADA activity is associated

with combined immunodeficiency and no evidence of hemolysis. Increased ADA activity,87 however, is associated with mild CNSHA. Red cell ATP concentra­tion is characteristically reduced to about 50 to 60 percent of normal.Conclusion

The previously discussed enzymopa­thies can cause chronic non-spherocytic hemolytic anemia (CNSHA) which may lead to anemia and jaundice in the new­born period. The use of enzyme screening tests for G-6-PD, PK, and GPI deficien­cies require small amounts of blood that may be obtained by heel puncture and are helpful in the diagnosis of the three most common enzymopathies of the HMP shunt and glycolytic pathway. However, a

negative screen does not definitely rule out PK or G-6-PD deficiency, as previ­ously discussed. Another more common enzymopathy, PD-5'-N deficiency, is char­acterized by basophilic stippling of the red cells. Analysis of the pattern of glyco­lytic intermediates may reveal a crossover site and suggest a specific enzymopathy. The 2,3-DPG concentration can be meas­ured with blood obtained from a heel puncture and may be decreased (suggest­ing an enzyme deficiency at or above the DPGM step) or increased (suggestive of an enzymopathy distal to DPGM).

In many instances, however, after ex­haustive investigation of known enzyme deficiencies in specialized laboratories, a specific enzyme deficiency may not be identified.

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