Hereditary Hemolytic Anemias Associated with Specific Erytbrocyte Enzymopathies

Medical Progress

WILLIAM N. VALENTINE, M.D., Los Angeles

IT IS INCREASINGLY apparent that the hereditary days and more than 100 miles of travel in the cir- hemolytic anemias of man comprise a vast and culation. Nonetheless, its energy requirements, variegated spectrum of diseases, some compara- while small, are still finite. It must maintain its tively common and some very rare, which have biconcave, discoidal shape, pump sodium out of genetically determined aberrations in either struc- and potassium into its intracellular environment tural or catalytic (enzyme) proteins of the erythro- against electrochemical gradients, protect itself cyte. In some, the inborn error results in the pres- from oxidative stresses in its surroundings, and ence of an abnormal hemoglobin; in some, such prevent significant conversion of its oxyhemoglobin as the thalassemic syndromes, inability to synthe- to methemoglobin. Under normal conditions, there size adequately one or another component of glob- is slow denaturation of its crucial metabolic pro- in appears to be the fundamental abnormality; in teins each of which, under any given set of circum- some, such as those forming the subject of this stances, has its individual biological half-life. Pre- review, specific enzyme deficiencies constitute the sumably its ultimate destruction results from attri- molecular basis of anemia. In still other instances, tion of its metabolic capacities and its eventual including hereditary spherocytosis, the precise eti- inability to provide even the austere energy re- ology at the molecular level remains undefined. quirements necessary for survival.1-5 The mature erythrocyte in man derives essen- Normal Erythrocyte Metabolism tially all of its energy requirements from the con- The mature human erythrocyte has lost much of version of glucose to lactate. Chart 1 indicates the the metabolic machinery characteristic of the other available routes of glucose metabolism. Glucose, nucleated cells of the body. It has no nucleus, no phosphorylated by hexokinase to glucose-6-phos- ribosomes, mitochondria or other intracellular or- phate (o-6-P), may traverse two main pathways. ganelles, only ineffectual vestiges of Kreb cycle The quantitatively predominant Embden-Meyer- metabolism, and no capacity for oxidative phos- hof pathway results in the anaerobic production of phorylation. Beyond the reticulocyte stage, it re- two moles of lactate for each mole of glucose me- tains no mechanism for synthesizing new protein. tabolized. In the first half of the cycle, triose is Some 95 percent of its dried weight is hemoglobin, formed from glucose, and energy is expended in which performs its crucial metabolic functions the form of the conversion of adenosine triphos- withput expenditure of energy. However, its simple phate (ATP) to adenosine diphosphate (ADP) at metabolic requirements are so geared to its re- the hexokinase and phosphofructokinase mediated stricted capabilities that it normally survives 120 steps. In the simplest illustration, this initial energy loss From the Deartment of Medicine, University of California Center for the Health Sciences, and Wadsworth Hospital, Veterans Adminis- is more than compensated by the production of tration Center, Los Angeles. Certain reported studies from the author's laboratory were supported four moles of ATP from one. mole of glucose (two by U.S.P.H.S. Grant HE 1069, by grants from Leukemia Research of triose) the terminal half of the path- Society, Los Angeles, and by the Louis B. Mayer Foundation for Medi- traversing cal Education and Research. way. Two moles of ATP are regenerated from ADP The author is Professor and Chairman, Department of Medicine, University of California School of Medicine, Los Angeles. at the phosphoglycerate kinase step, and two more Reprint requests to: Department of Medicine, UCLA Center for the Health Sciences, Los Angeles 90024. at the point of the pyruvate kinase mediated reac-

280 APRIL 1968 *. 108 * 4 don. The net result is a gain of two moles of high versing the energy wasteful Rapaport-Luebering energy ATP from each mole of anaerobically metab- shunt6'7 shown in Chart 1. The latter is a unique olized glucose. This net gain, however, may be feature of the erythrocytes of man and certain, but materially reduced if triose by-passes the ATP gen- not all, mammalian species. The glycolytic inter- erating phosphoglyceratekinase reaction by tra- mediate, 1,3-diphosphoglycerate (1,3-DPG) may Glucose ATP H/EXOK/N/ASE jpq Mg* GLUCOSE-6-PHOSPHArE ADP DEHYDROGENASE G-6-P-a /" *No6-PG GLUCOSEPHOSPHA TE ISOMERASE NADP NADPH PSPHOGLUCONATE F-6-P GSH NADP DEHfVYDROGENASE ArP SE' PHOSPHOFRUCTOKINASE A mg LurATrH/ONE' RREDUCrA ADP ' \ GSSG JNADPH) F-I,6-P \ FCO2 FRUCTOSEDIPHOSPHATE ALDOLASE T TO TRANSKETOLASE \ v se-5-P DHAP \ TRANSALDOLASE .~~~~Pentos TRIOSEPPHOSPHATE /SOMERASEN ,/ G-3-P GLYCERALDEHYDE-3-PHOSPHATE PI/ NAD DEHYDROGENASE DH 3/ 193-DPG 1,3- D/PHOSPHOGL Y CEROMUTASE ADP4 PHOSPHOGLYCERATE K/NASE J Mg0 2,3-DPG A TP - 3-PG DIPHOSPHOGLYCERATE PHOSPHATASE PHOSPHOGLYCEROMUTASE t,3-DPG 2-PG PHOSPHOPYRUVATE HYDRATASE M9r PEP PYRUVATE KINASE ADPS Mg9 ATP K* Pyruvate NADH LACTATE DEHYDROGENASE NAD Lactate Chart 1.-The pathways of erythrocyte glycolysis.

CALIFORNIA MEDICINE 281 be mutated enzymatically to 2,3-diphosphoglycer- this manner is again available to participate in the ate (2,3-DPG), and the latter may accumulate to metabolism of the HMP oxidative shunt. the point where at times it contains some 50 per- While quantitatively less active than the anaero- cent of the total erythrocyte organic phosphorus.8 bic cycle under normal conditions, the HMP shunt It, in turn, may be dephosphorylated and returned is greatly stimulated by oxidative stresses resulting to the mainstream of glycolysis as 3-phosphoglyc- in glutathione oxidation, and NADPH, generated by erate (3-PG), which is also the product of the al- the shunt here permits the important glutathione ternative phosphoglycerate, kinase reaction of the reductase reaction to proceed. This constitutes a anaerobic pathway. protective mechanism invoked, for example, in the It is apparent that if glucose were entirely me- face of the challenge of oxidative damage resulting tabolized via 2,3-DPG-that is, through the Rap- from the administration of certain drugs. Under aport-Luebering shunt-two moles of ATP would such conditions, the operation of the HMP shunt be lost before the formation of triose, and only two may be crucial to cell survival. In view of the moles would be later generated (at the pyruvate highly restricted metabolic resources of the eryth- kinase step only). No net gain would result from rocyte, it is not surprising that a variety of highly this exercise in futility. Therefore, in considering specific and sharply localized inborn metabolic the strictly anaerobic erythrocyte metabolism of a errors should result in impaired survival and, mole of glucose, a net gain of somewhere between hence, in hemolytic anemia. 0 and 2 moles of ATP winl result, depending on what proportion of glucose passes through the Hemolytic Anemia and 2,3-DPG pool. The latter may be important in regu- Erythrocyte Enzyme Deficiency lating intracellular ATP/ADP ratios and level of The ultimate definition of the molecular etiology inorganic phosphorus, and may also serve as a of the now recognized enzyme deficiency associated reservoir of substrate when glucose is temporarily hemolytic anemias received major impetus from depleted in the environment of the cell. In the an- two main sources. The first of these resulted from aerobic cycle, the pyridine co-factor is diphospho- a revival of interest in the known induction of se- pyridine nucleotide (NAD) and this is cyclically vere hemolytic episodes in a significant proportion reduced at the triosephosphate dehydrogenase step of persons receiving antimalarial agents such as to NADH and reoxidized in the course of the termi- primaquine.10 Extensive studies during and after nal lactate dehydrogenase reaction. World War II culminated in 195611 in the recogni- While normally some 90 percent of glucose is tion that "primaquine sensitivity" was conditioned thought to traverse the anaerobic route,9 a signifi- by a specific deficiency of glucose-6-phosphate de- cant and highly variable portion of glucose is me- hydrogenase (G-6-PD), the first enzyme of the tabolized via the so-called hexose monophosphate HMP shunt pathway. For the first time the mech- (HMP) oxidative shunt, likewise shown in Chart 1. anism of an adverse reaction to a drug, whose ad- Glucose-6-phosphate oxidized by this alternative ministration was benign in most but catastrophic pathway is converted to pentose through the action in a few, was defined at the molecular level and of two dehydrogenases. Pentose so generated is, shown not to be on an immunological basis. through a series of enzymatically catalyzed reac- The second impetus derived from the studies tions, again converted to fructose-6-phosphate of Dacie and his colleagues12 in England. These (F-6-P) and glyceraldehyde-3-P (o-3-p), inter- workers were interested in certain relatively un- mediates of the Embden-Meyerhof pathway which common hemolytic anemias which they character- ultimately become lactate. It should be noted that ized as "nonspherocytic," in contrast to the better triphosphopyridine nucleotide (NADP) is the co- known disorder of hereditary spherocytosis. This factor operative in the oxidative shunt. It is re- heterogeneous group of hereditary anemias pos- duced to NADPH by the first two dehydrogenase sessed certain clinical and laboratory features in reactions, during which CO2 is evolved and pentose common. No hemoglobinopathy was demonstrable, formed. NADPH is reoxidized by enzymatic reac- the antiglobulin (Coombs) test was negative, the tions outside of the strictly glycolytic metabolism. osmotic fragility of fresh cells was usually normal, The most important of these, catalyzed by NADPH- splenomegaly was the rule, and splenectomy (un- linked glutathione reductase, maintains glutathione like the situation in hereditary spherocytosis) at (GSH) in its reduced form. NADP regenerated in best ameliorated, but did not clinically correct,

282 APRIL 1968 * 108 * 4 the disorder. Studies of glucose metabolism in the ceptibility to primaquine hemolysis but not with reticulocyte-rich, young erythrocyte populations chronic anemia. Harvald and his associates24 have of affected subjects in some instances suggested reported hereditary hemolytic anemia in two young relatively deficient glycolysis and raised the ques- men with a deficiency of adenosine triphosphatase tion of some form of metabolic impairment in su- (ATPase). This was regarded as a probable cause gar utilization. of the anemia. Necheles and coworkers25 have re- Selwyn and Daciel" devised an in vitro "autohe- cently described a relatively severe deficiency of molysis" test whereby sterile, defibrinated blood, the enzyme glutathione peroxidase in a male with and without additives such as glucose, was in- subject who exhibited a hemolytic reaction after cubated at 37°C for 48 hours and spontaneous transfusion, and a probable mild, compensated hemolysis was quantitated. Different patterns were hemolytic disorder after recovery. Glutathione per- observed among the cell populations from differ- oxidase catalyzes the oxidation of glutathione by ent subjects studied. In one, categorized as Type peroxides, and evidence suggests it is of importance I, autohemolysis was only slightly greater than in in protecting cellular proteins from peroxidative normal blood, and was nearly, but not quite, cor- denaturation. Oort, Loos and Prins and their col- rectible by adding glucose during the incubation. leagues26-28 have extensively studied a kindred in In a second, Type II, autohemolysis was usually which certain members have a moderately severe severe, and was poorly if at all corrected by addi- hemolytic process and nearly complete absence of tives of glucose or adenosine. The test, although erythrocyte glutathione. The inborn error is since shown to lack critical specificity, suggested thought to be inability to fully synthesize the tri- the heterogeneity of these hereditary disorders. A peptide glutathione. little later de Gruchy and his colleaguesl4-6 in Aus- (3) Deficiencies in enzymes of the anaerobic tralia measured glycolytic intermediate levels in the glycolytic pathway. Hemolytic disorders associated erythrocytes of similar cases. In certain instances with inherited deficiencies of the enzymes pyruvate an abnormal accumulation of intermediates sug- kinase (PK),17,1819,29,30 triosephosphate isomerase gested "pile-up" behind a point of metabolic block, (TPI),31-33 hexokinase (HK) 3435 glucosephosphate which appeared to be distal to triose formation. isomerase (GPI) ,36,37 phosphoglycerate kinase Again, a defect in the glycolytic machinery was (PGK),38a-b and of 2, 3-diphosphoglyceromutase suggested. In 1961 the first of several genetically (2, 3-DPGM)39-42 are now established. determined erythrocyte glycolytic enzyme deficiencies associated with chronic hemolysis-that of HMP Oxidative Shunt Pathway pyruvate kinase deficiency-was defined by Valen- Enzyme Deficiencies and Miwa.17"18 tine, Tanaka G-6-PD Deficiency Classification (Table 1) The fact that the antimalarial agent pamaquine At present a number of hemolytic anemias in is capable of causing hemolytic episodes in cer- which the inborn error has been identified are rec- tain persons had been known since 1926.43 In ognized. These enzymopathies may be categorized 1948 Earle and associates," noted that about 10 as follows: percent of American Negroes were susceptible, ( 1 ) Enzymatic deficiencies in the oxidative HMP while hemolysis was relatively rare in Caucasians shunt pathway. G-6-PD deficiency has been widely in this study. Similar findings were reported for studied.'0'19 A few reports20'2' of possible associa- primaquine by Hockwald and co-workers45 in tion of hereditary hemolytic anemia with partial 6 1952, and in 1954 Dern and co-workers46 clearly phosphogluconate dehydrogenase (6-PGD) de- demonstrated an intrinsic erythrocyte abnormality ficiency are available, but a cause-effect relation- in susceptible subjects. Chromium-labeled erythro- ship here is uncertain. cytes from patients with "primaquine sensitivity" (2) Enzymatic deficiencies in non-glycolytic were rapidly destroyed after transfusion into enzymes. Chronic hemolytic anemia associated normal persons when primaquine was given to the with erythrocyte glutathione reductase (GSSG-R) recipient. Conversely, normal erythrocytes, isotopi- deficiency has been observed by Lohr and Waller.22 cally labeled and transfused into primaquine-sen- Carson and co-workers23 have reported a milder sitive subjects, exhibited unimpaired survival when deficiency of the same enzyme associated with sus- the drug was subsequently administered.

CALIFORNIA MEDICINE 283 TABLE 1.-Erythrocyte Enzymopathies Associated with Hereditary Henolytic Anemia Delicient Enzyme Reaction Catlyzed A. HEXOSEMONOPHOSPHATE-SHUNT ENZYME DEFICIENCIES G-6-PD 1. Glucose-6-phosphate dehydrogenase: G-6-P + NADP -4 6-PG + NADPH 6-PGD 2. 6-Phosphogluconate dehydrogenase: 6-PG + NADP P Ru-S-P + C02 + NADPH B. NONGLYCOLYTIC-ENZYME DEFICIENCIES GSsG-R 1. Glutathione reductase: GSSG + 2 NADPH 4_ 2 GSH + 2 NADP ATP-ase 2. Adenosine triphosphatase: ATP - 0 ADP + Pi Na+ K+ Mg' GSH-PX 3. Glutathione peroxidase: 2 GSH + H202 GGSSO + 2 H20 GS-I 4. Glutathione synthetase: (a) Glu + Cyst o- y-Glutamyl Cyst ATP Mg++ + 2 ADP + 2 Pi GS-II

(b) y-Glutamyl Cyst + Gly - GSH ATP Mg++ C. GLYCOLYTIC-ENZYME DEFICIENCIES PK 1. Pyruvate kinase: PEP + ADP -4-- Pyruvate + ATP K+ Mg++ TPI 2. Triosephosphate isomerase: DHAP -4* G-3-P HK 3. Hexokinase: Glucose + ATP o-6-PG + ADP Mg++ GPI 4. Glucosephosphate isomerase: G-6-P F-6-P PGK 5. Phosphoglycerate kinase: 1,3-DPG + ADP -4- 3-po + ATP Mg++ DPGM 6. Diphosphoglyceromutase: 1,3-DPo - P 2,3-DPG 3-PG Abbreviasions: G-6-P = glucose-6-phosphate, 6-PG = 6-phospho-3-keto gluconate, Ru-5-P = ribulose-5-phosphate, PEP = phosphoenol pyruvate, D"AP = dihydroxyacetonephosphate, G-3-P = glyceraldehyde-3-phosphate, F-6-P = fructose-6-phosphate, 1,3-DGP = 1,3-diphosphoglycerate 2,3-DGP = 2.3-diphosphoglycerate, 3-PG = 3-phosphoglycerate, NADP = nicotinamide-adenine dinucleotde phosphate (TPN), NADPH = reduced nicotinamide-adenine dinudeotide phosphate (TPNH), ATP = adenosine triphosphate, ADP = adenosine diphos1phate, H202 = hydrogen peroxide, GSSG = oxidized glutathione, GSH = reduced glutathione, Cyst = cysteine, Gly = glycine, Glu = glutamic acid, Pi = inorganic phosphorus.

The clinical events of the hemolytic syndrome tate hemolysis in like manner in sensitive subjects. were later defined in detail by Dern, Beutler and In 1956, Carson and his associates"1 demon- Alving.47 Beutler then demonstrated that the strated that the underlying defect was an inherited erythrocytes of affected persons had a low content deficiency in the enzyme glucose-6-phosphate de- of glutathione, and that this rapidly declined fur- hydrogenase. The same erythrocyte abnormality ther when primaquine was administered.'" Further, was subsequently shown to exist in all subjects sus- in vitro incubation of primaquine-sensitive erythro- ceptible to a hemolytic syndrome induced by the cytes with drugs such as acetylphenylhydrazine ingestion of fava beans or the inhalation of pollen resulted in pronounced and rapid disappearance of from flowering fava bean plants.52-55 Some subjects erythrocyte-reduced glutathione49 and an abnor- with c-6-PD deficiency can apparently eat fava mality in Heinz body production.50 Heinz bodies beans with impunity,56'57 and some factor other are intracorpuscular inclusions, demonstrable by than enzyme deficiency appears required. Hemo- supravital staining with methyl violet or brilliant lytic susceptibility after fava bean ingestion is cresyl blue, and represent oxidatively injured and almost exclusively limited to the non-Negro type denatured hemoglobin.51 Many drugs,10 including of deficiency. sulfonamides, acetanalid, furadantin, and certain It is now known that about 2 percent of subjects vitamin K derivatives, were soon found to precipi- with 0-6-PD deficiency have a chronic hemolytic

284 APRIL 1968 * 108 * 4 anemia in the absence of drug challenge,58-60 and resents the length of time necessary to restore mean that infectious processes may53 also cause hemoly- cell age to the pre-hemolytic status. Severity of sis to some degree. hemolysis is dose related and drug blood level G-6-PD deficiency is thought to affect some 100 related, liver and kidney impairment being capa- million people in a geographic distribution similar ble of affecting the latter. For more extensive and to that of falciparum malaria.61'62 Among Cauca- detailed discussions, the reader is referred to avail- sians it is common in Sardinians and in Sephardic able reviews.10 or Oriental, but not in Askinazy or European, Jews; but the disorder has been found in almost 6-Phosphogluconate Dehydrogenase (6-PGD) all racial groups. About 11 percent of American Deficiency Negroes exhibit the deficiency. Extensive studies Brewer and Derm75 and Parr and Fitch76 have have documented genetic polymorphism63-67; that reported partial deficiency of 6-PGD in the erythro- is, many genetic variants, the result presumably cytes of certain persons without hemolytic anemia. of different mutations at the same locus, have now The enzyme activity was of the order of 50 percent been observed and studied. For example, G-6-PD of normal. In contrast, two cases of hereditary deficiency is readily demonstrable in the leuko- hemolytic anemia (those of Lauseckher and co- cytes of affected Caucasians," but this is much workers,20 and of Scialom and colleagues2' have less evident in Negroes. The disorder is X chromo- been reported in which 6-PGD was reduced to about some-linked.10'69-7l Males and homozygous females the same degree. It is, however, entirely possible have full expression of the abnormality and severe that this moderate reduction was not related to deficiency; heterozygous females have wide vari- the hemolytic process causally, and may have co- ability in the degree of enzyme deficiency, and in incidentally been present with some other undetect- the severity of hemolytic episodes. The enzyme i ed etiologic factor. erythrocytes may be quantitatively assayed and a spot test72 and tests based on the diminished ca- Non-glycolytic Enzyme Deficiencies pacity of affected erythrocytes to reduce certain Glutathione Reductase (GSSG-R) Deficiency dyes61'73 or methemoglobin are also available for Lohr and Waller22 have reported a kindred, use in the clinical laboratory or in survey studies certain members of which had chronic hemolytic in the field. anemia associated with a relatively severe erythro- The mechanism of drug induced hemolysis in cyte deficiency in the enzyme glutathione re- y-6-PD deficiency is thought to reside in the in- ductase. Waller and associates77 have also reported ability of affected cells to generate the necessary hematologic and neurologic difficulties associated NADPH to maintain glutathione in its reduced with GSSG-R deficiency. Carson and co-workers23 form.10 This results in impaired protection against have likewise noted susceptibilty to primaquine a number of noxious oxidative challenges. hemolysis, but not chronic anemia, in a much less Clinically,10'47 acute hemolytic anemia, jaundice severely deficient subject. Glutathione reductase and hemoglobinuria develop on the second to reduces oxidized glutathione and concomitantly fourth day after ingestion of an invoking drug. oxidizes NADPH. It interacts with the dehydro- Only the older cells are destroyed, but in severe genases of the oxidative shunt to protect against instances as many as 30 to 50 percent of circulat- noxious oxidative processes and to maintain the ing cells may be hemolysed. Because hemolytic level of reduced glutathione. In sufficiently de- susceptibility is related to erythrocyte age, recovery ficient states this protective mechanism is impaired. usually takes place beginning at about the tenth day or a little later, even if the drug is continued. Adenosine Triphosphatase (ATPase) Deficiency With continued drug administration, an equilib- A sodium-potassium-magnesium dependent rium phase occurs in which there may be no ouabain inhibitable ATpase is generally considered anemia, and in which hemolysis referable to the to play a central role in the active transport of destruction of cells attaining a critical age is de- sodium and potassium across cell membranes. tectable only by special erythrocyte survival Harvald and his associates24 have briefly reported studies. After discontinuance of the drug, about three unrelated persons, two of whom had chronic, three or four months must elapse before an episode apparently inherited, hemolytic anemia, in whom of comparable severity can be reinduced. This rep- ATpase levels in the red cell approximated half-

CALIFORNIA MEDICINE 285 normal values. These workers consider it likely bated with normal cells radioactive GSH was that ATpase deficiency and the chronic hemolytic formed. When similar incubations were performed process were related. Family studies suggested a with the affected cells no radioactive GSH could be possible dominant inheritance, but normal ATpase found. Breakdown of added radioactive oxidized levels in the cells of both parents of one propositus glutathione occurred at an equal rate in GSH de- did not conform to this hypothesis. The issue was ficient and normal cells. It was concluded that a further clouded by uncertainty as to whether the defect in GSH synthesis was probably involved. It transport related ATPase or, perhaps, other forms is remarkable that such low levels of erythrocyte of ATPase activity as well were involved. Additional GSH are compatible with survival, but such has data are required before these questions can be been clearly demonstrated to be the case. resolved. Deficiencies in Embden-Meyerhof Glutathione Peroxidase Deficiency Pathway Enzymes The presence of glutathione peroxidase (GSH- Pyruvate Kinase (PK) Deficiency Px) in mammalian erythrocytes was first estab- In 1961 and 1962 erythrocytes from certain lished by Mills.78'79 The enzyme catalyzes the in cases of hemolytic anemia, conforming in terms vitro detoxification of hydrogen peroxide. It there- of the autohemolysis test to the Type II category fore provides protection against peroxidative in- of Selwyn and Dacie, were assayed for all glycolytic jury, and is linked to HMP shunt pathway meta- enzyme activities. All except one - pyruvate bolism through its role in glutathione oxidation. kinase, the enzyme converting phosphoenol pyru- A partial deficiency in GSH-Px has been described vate to pyruvate and located just above the ter- in association with transient mild.hemolysis in the minal lactate dehydrogenase step - were found to neonatal period.80 Necheles and his associates25 have normal or greater than normal activity.17'18 have recently described an apparently homozygous Chart 2 indicates the pyruvate kinase reaction and state of the enzymopathy in a Puerto Rican male the essentials of the assay system. in whom a hemolytic reaction developed after Since that time about a hundred documented transfusion. After recovery, mild reticulocytosis cases of hemolytic anemia with hereditary de- suggested persistence of a compensated hemolytic ficiency of this enzyme have appeared in the world process. The cells of the patient possessed GSH-PX literature, and it appears thus far that, except for activity about 25 percent of normal. The red cells of both parents exhibited about half-normal GSH-PX activity. A recessive mode of inheritance was sug- PEP ADP gested. In studies with isotopically labelled glucose, hydrogen peroxide decidedly stimulated HMP shunt Mg activity in normal subjects, but did not do so in PYRUVATE K/NASE (Lysote) the patient. While it is presumed this would result in increased susceptibility to drug induced oxidative injury, data on this are thus far unreported. Pyruvate ATP Glutathione (GSH) Deficiency 4 A series of studies in a Dutch2628 kindred have ev NADH A340 mri demonstrated moderately severe, chronic, heredi- Lactate Dehydrogenase tary hemolytic anemia associated with a remark- (Crystalline) able and nearly total absence of either oxidized or K. NAD reduced glutathione. In intact erythrocytes, gly- oxalase activity, for which GSH is an obligatory co- Lactate factor, was zero. As expected, the affected cells Chart 2.-Diagrammatic representation of the principles were primaquine-sensitive and, in addition, chrom- of the pyruvate kinase (PK) assay system. Substrate PEP in shor- (phosphoenol pyruvate) is provided in buffer containing ium, even labeling amounts, remarkably ADP, Mg++, and K+. A dilute hemolysate provides the PK tened cell survival in vivo. When radioactive gly- being assayed. Pyruvate generated is converted to lactate cine, glutamine, and cysteine, the amino acid com- in the presence of added lactic dehydrogenase and the concomitant oxidation of NADH is followed spectrophoto. ponents of the tripeptide glutathione, were incu- metrically.

286 APRIL 1968 * 108 * 4 c-6-PD deficiency, this is the commonest heritable even hexoses behind the point of metabolic disorder in the enzyme deficiency group.29'86 Cases block.84'89'91-93'95 In such cases, pyruvate kinase, are of variable severity. Some patients may reach which ordinarily operates well below substrate adult life partially compensated, with variable saturation in the cell,96 is rendered more effective reticulocytosis and anemia and escaping transfu- by the greater availability of substrate. This com- sion. Others require transfusion from infancy and pensates to some extent for the low enzyme activity are unable to survive without donated blood. Most characteristic of the disorder. As a result, young exhibit macrocytosis, a morphologically nonde- cell populations of homozygotes may still glycolyse script red cell with a scattering of cells with ir- actively and reasonably effectively maintain ATP regular membrane borders resembling acantho- levels. In some instances, defective ATP mainte- cyctes, splenomegaly and iron-containing inclu- nance can be demonstrated by in vitro incubations, sions in erythrocytes (Pappenheimer bodies) only and similar studies may show inability to cycle if they have undergone splenectomy.'8 NAD to NADH and back adequately. In certain cases, Splenectomy only ameliorates the hemolytic however, defective ATP maintenance and pro- process, and it was our original view that it was nounced intermediate accumulation may not be of little benefit. Accumulated experience8la has demonstrable.30'97,98 indicated, however, that removal of the spleen may It must be remembered that only metabolically be accompanied by significant improvement, some- affluent young cells, still capable of compensating times with lessening or disappearance of transfu- for their glycolytic deficiency, are available for sion requirements. In the severe cases of Bow- assay. As the cell ages, however, increasing accu- man8lb182 in an Amish population it appears that mulation of intermediates proximal to the deficient splenectomy is necessary for survival. Now we enzyme and eventual inability to generate ATP and believe that splenectomy is often of distinct value cycle NAD are to be expected. and indicated, particularly when transfusions are In most cases, the kinetics of residual enzyme required or anemia is significantly symptomatic. activity in homozygotes has been normal. Again, In a few cases, red cells have been morphologically such has not been universally true, and genetic bizarre,83-85 with most cells showing numerous ir- polymorphism in the case of pyruvate kinase de- regular projections of the membrane surface. ficiency, as in G-6-PD deficiency, is becoming in- Anemia is exacerbated by surgical stress and inter- creasingly apparent. Instances of pyruvate kinase current infections. deficiency and abnormal enzyme kinetics have In the commonest situation, when pyruvate been reported by Waller and L6hr,95 by Boivin and kinase activity is assayed its activity is found to be Galand,99 and by Miwa.'00 Hsu, Robinson and greatly reduced and occasionally undetectable Zuelzer98 have intensively studied a kindred in (though, of course, not absent) in deficient which apparent homozygosity for pyruvate kinase erythrocytes. Both sexes are affected, and both activity existed in certain members with sharply parents, all offspring and a number of the siblings differing clinical symptomatology and glycolytic and relatives of symptomatic subjects have on the intermediate patterns. In some members the char- average about one-half normal pyruvate kinase acteristic hemolytic syndrome was present. In activity in their red cells. The disorder, therefore, others, equally severely affected in terms of enzyme appears to be transmitted as an autosomal reces- assay levels, there were no symptoms and, at most, sive trait, heterozygotes being clinically and a mild, fully compensated hemolytic process. The hematologically normal but biochemically detect- coexistence of two genes, undifferentiable as to able, and homozygotes having symptomatic hemo- their effect on pyruvate kinase in the usual assay, lytic anemia.'7'18 The leukocyte pyruvate kinase but with decidedly different clinical expression, was activity is normal.17"18'87,88 As would be expected postulated. with a block in terminal glycolysis, deficient cells In our laboratory, four cases of severe hereditary in many instances may exhibit suboptimal gly- hemolytic anemia in two unrelated families with colysis for their age,'3'30'86 may have difficulty in clinical and biochemical features of erythrocyte py- maintaining ATP13 83'893 and NAD,3089'93'94 and as ruvate kinase deficiency, but with assay values in say of glycolytic intermediates frequently reveals either the normal or high heterozygote range have abnormal accumulations of phosphoenol pyruvate, recently been studied by Paglia and coworkers.'0' triose, particularly 2, 3-diphosphoglycerate, and Cases with similar intermediate or normal PK assay

CALIFORNIA MEDICINE 287 values, but with the usual picture of homozygous served in the erythrocytes of pyruvate kinase de- deficiency have been noted by others.86'88'94,lW1"02.103 ficiency, the total syndrome, clinically and bio- In both families, the apparent dilemma was re- chemically, presents many differences:31,32,33a,33b solved by the finding that affected subjects had * The autohemolysis test resembles that seen in inherited from one side the "usual" gene resulting hereditary spherocytosis. This is to say that sub- in pyruvate kinase deficiency, and from the other stantial autohemolysis, well corrected by additives a gene resulting in a pyruvate kinase with grossly of glucose and adenosine, is observed.31 aberrant kinetics. The latter was characterized by remarkable catalytic inefficiency at low concentra- * Dihydroxyacetone accumulation is a promi- tions of its substrate, phosphoenol pyruvate, such nent biochemical feature.33a,33b as exist within the metabolizing erythrocyte. At * The enzyme deficiency is present in leuko- high substrate concentrations (such as those em- cytes, muscle and cerebrospinal filuid33a and pos- ployed for convenience in the usual assay) essen- sibly (though this is not yet certain) in other body tially no difference was evident. Heterozygosity for tissues. the kinetically abnormal gene was traceable by * An atypical, severe and progressive neuro- kinetic studies through one side of three genera- logic deficit, involving peripheral nerves and cen- tions in each of the two families. Not only was the tral nervous tissue, has been noted in the small Km for the abnormal pyruvate kinase some ten number of affected children who have survived times greater than that of the normal enzyme, but the first few months of life.31,32,33a,104 This is clearly also differences in pH optimum and heat and stor- not related to kernicterus, and quite possibly may age stability were readily demonstrable. Heterozy- be the clinical manifestation of the isomerase de- gotes had enzyme kinetics intermediate between ficiency in nervous tissue. normal subjects and the affected propositi. * Two unexplained sudden deaths in affected The syndrome of pyruvate kinase deficiency children have led to clinical suspicion (thus far hereditary hemolytic anemia may thus clearly re- unconfirmed) that cardiac muscle dysfunction may sult from a quantitative catalytic deficiency or, as at times be a significant feature.33a in the above cases, from a qualitative defect of the same enzyme. With further studies it is likely that It is of tangential interest that sickle cell trait additional evidence for genetic polymorphism will and G-6-PD deficiency have been demonstrated in be forthcoming. Sachs and coworkersl03 have like- the same kindreds, with a variety of combinations wise reported pyruvate kinase deficient hemolytic of these inherited metabolic errors and TPI defi- anemia in association with an enzyme catalyti- ciency.32 Again, in TPI deficiency both sexes are cally inefficient at low substrate concentrations. involved, heterozygotes and homozygotes are de- finable, and an autosomal recessive mode of in- Triosephosphate Isomerase (TPI) Deficiency: heritance is indicated.31'32'104 In 196531,104 and 196632 another higbly con- sanguineous kindred of mixed Caucasian and Ne- Hexokinase (HK) Deficiency: gro ancestry was studied. In members with hemo- A single kindred has been extensively studied lytic anemia resembling that of pyruvate kinase in which one child has had hemolytic anemia since deficiency, a separate deficiency-that of triose- birth, and in which erythrocyte, but not leukocyte, phosphate isomerase-was demonstrated. In the hexokinase activity is extremely low.34105 Erythro- process of anaerobic glycolysis, aldolase converts cyte hexokinase activity must be evaluated in the hexose diphosphate into two triose isomers, 3- context of the fact that of all the glycolytic en- phosphoglyceraldehyde and dihydroxyacetone zymes it exhibits by far the greatest increased ac- phosphate. These are interconverted by the iso- tivity in reticulocytes and young erythrocytes. merase deficient in the erythrocytes of these sub- Hexokinase activity in reticulocyte-rich cell popu- jects. lations is severalfold-up to ten times or more- The very small number of subjects thus far greater than in populations of mixed age in normal studied, with the exception of one apparently un- blood. In the case of the propositus, despite pro- related case, derive from a population group with nounced reticulocytosis and a very young mean a high degree of consanguinity. While morpho- erythrocyte age, red cell hexokinase activity was logically the findings are very similar to those ob- at the lower limit of normal, and six to eight times

288 APRIL 1968 * 108 * 4 less than in simultaneously assayed populations of trophoretically different GPI protein, have been in- comparably young control blood. Morphologically, herited by the propositus. Again, additional studies the hemolytic anemia was essentially similar to that are required. observed in other erythrocyte enzyme deficiency states. Autohemolysis was modestly increased, par- Phosphoglycerate Kinase (PGK) Deficiency: tially corrected by glucose and better corrected by We3sa.38b have recently had the opportunity of adenosine. Both glucose and fructose were phos- studying in conjunction with colleagues from other phorylated at low rates, but mannose and galactose laboratories the erythrocytes and leukocytes of a were metabolized much more rapidly. In both Chinese boy with lifelong hemolytic anemia. Both parents and three siblings, values for hexokinase red and white cells exhibited severe deficiency of were either distinctly low or at the extreme lower PGK. The hematological findings were essentially limits of the normal range, suggesting heterozy- identical with those observed in other glycolytic gosity for the deficiency and, again, an autosomal enzyme deficiencies. There was evidence of mental recessive mode of inheritance. Presumably the retardation and behavioral aberrations, but it is patient's erythrocytes, already hexokinase-deficient not known whether or not these are related to the in youth, with maturation and further attrition of enzymopathy. Thus far, analysis of glycolytic in- the already impaired enzyme, soon are rendered termediates is incomplete. The case is of particu- incapable of survival. lar interest since PGK activity was severely reduced to an estimated 4 to 5 percent of normal. The abil- Glucosephosphate Isomerase (GPI) Deficiency: ity of erythrocytes to glycolyze and survive in these Four children in two unrelated kindreds36'37,106 circumstances implies extensive utilization of the have now been shown to have severe hereditary Rapaport-Luebering shunt or of unknown meta- hemolytic anemia associated with a deficiency of bolic pathways. The genetics are at present ob- glucosephosphate isomerase, the second enzyme scure. The mother of the patient has a mild, com- of the anaerobic glycolytic pathway, catalyzing the pensated hemolytic process with reticulocytosis of interconversion of the isomers glucose-6-phosphate 7 to 10 percent and suggestively low activity of and fructose-6-phosphate. Leukocytes in all four PGK. The possibility of X-chromosome linkage is children share the deficiency. Other body tissues under consideration but unproved. have not been adequately studied but, if affected, Very recently Kraus and coworkers38a reported no evidence of clinical dysfunction exists. The an apparently similar case, characterized by propositi all exhibit a severe hemolytic syndrome. chronic hemolytic anemia and PGK deficiency most Splenectomy has proved of possibly some benefit, evident in older erythrocytes separated by density though pronounced reticulocytosis and hemolysis gradient techniques. The patient had no known persist. living relatives and genetic data could not be Again, both parents in each family and certain obtained. other family members have clearly intermediate GPI activity in their erythrocytes, and an autoso- 2,3-Diphosphoglycerate Mutase (2,3-DPGM) De- mal recessive inheritance pattern again pertains. ficiency: Studies with isotopically labeled glucose indicate Bowdler and Prankerdl08 have reported an in- striking impairment of recycling of fructose-6- ferred 2,3-DPGM deficiency in association with phosphate, derived initially from pentose formed hereditary hemolytic anemia. The inference was via the oxidative shunt, back through the partially based on finding about 50 percent normal 2,3- blocked isomerase reaction to glucose-6-phosphate DPGM in erythrocytes, a failure of 2,3-DPG to in- and thence again into the HMP shunt pathway. crease when glycolysis was inhibited in vitro with Starch gel electrophoretic studies,107 interestingly, sodium fluoride, and very small increases of 2,3- suggest that in the first case studied, the maternal DPG after incubations with glucose and inosine. The and paternal enzyme patterns are not identical. latter two findings were in contrast to those ob- Since both possess apparent heterozygosity for served with normal cells. glucosephosphate isomerase deficiency, it may be LUhr and Waller39 made similar inferences in that two separate mutated genes, both resulting in subjects with decreased 2,3-DPG content of ery- quantitatively deficient isomerase activity but elec- throcytes. The mutase itself was not measured di-

CALIFORNIA MEDICINE 289 rectly. The indirect measurements upon which in- The answers are at present imprecise and inad- ferences are based leads to some reservations as equate. First, though, it must be recognized that to the conclusive nature of the findings. a priori only those reticulocytes and young ery- Schrdter4l'42 presented more convincing obser- throcytes still capable of survival in the patient are vations in a child with very severe nonspherocytic available for study, and all metabolic parameters hemolytic anemia who died at age 3 months. Cu- measured are mean values for this cell population. riously, though, despite the evidence of a fulminat- The metabolic capabilities of these young erythro- ing hemolytic process, the reticulocyte count was cytes cannot be projected to define the metabolic only slightly elevated, and serum bilirubin only in status which hypothetically would exist at some the neighborhood of one milligram percent. The later stage of aging. Reticulocytes and young ery- child required almost daily transfusions and do- throcytes possess many metabolic advantages nated cells were also destroyed at a phenomenal which are lost with increasing maturity. They are rate despite inability to demonstrate any extra- able to prime the metabolic pipeline with glycolytic corpuscular mode of destruction. The constant subtrates much more effectively than can older presence of many foreign, homologous red cells cells as a result of the relatively great activity of in the infant's blood prevented meaningful enzyme many of their glycolytic enzymes and, particularly, assays in the child. However, 2,3-DPGM activity but not exclusively, hexokinase.5'34'5 The reticu- of the order of 50 percent of normal was demon- locyte still possesses Krebs cycle activity, the ca- strated by direct methods in both parents, a pa- pacity for oxidative phosphorylation and the abil- ternal sister and a grandmother of the propositus. ity to generate ATP by mechanisms other than those The results suggest that the propositus was homo- relating to the conversion of glucose to lactate. zygous for 2,3-DPGM deficiency, while the pheno- The metabolic impairment inherent in a severe typically healthy parents and affected relatives enzymopathy is, while severe, partial and never were heterozygous. The peculiar lack of more than complete. Compensatory mechanisms may in slight reticulocytosis or bilirubinemia, and the young cells, then, still permit survival. But a form rapid destruction of transfused cells remain an of selective metabolic progeria exists. Enzymati- enigma, however. cally, in a sense, such erythrocytes are old before their time, and normal survival is not to be ex- Discussion pected. The presence of highly specific erythrocyte en- To determine precise preludes to destruction re- zymopathies, genetically transmitted as either au- quires knowledge of the metabolic parameters of tosomal recessive or X chromosome-linked traits the older, moribund cell. Unfortunately, for ob- in a wide variety of the comparatively uncommon vious reasons, these crucial measurements cannot lifelong hemolytic anemias lends strong support be made. Further, the deleterious effects of accu- to a direct cause and effect relationship. This is mulated intermediates by feedback mechanisms rendered still more likely by the glycolysis-related are inadequately known and currently difficult to} nature of observed deficiencies and the established ascertain. In addition, far too little is known about unique dependence of the metabolically impover- qualitative and kinetic differences in the catalytic ished erythrocytes upon the glycolytic machinery. proteins deficient in these hemolytic states. It is Still, certain unresolved questions clearly exist. If clear that genetic polymorphism is in some in- the enzymopathies interfere with glucose metab- stances present, and that qualitatively different en- olism, why do in vitro studies at times indicate zyme molecules can vary, not only in catalytic ca- reasonably adequate glycolysis and ATP mainte- pacity in a given assay procedure, but also in nance in the cells of affected subjects? Why are susceptibility to denaturation with aging and in sharp phenotypic differences observed in the clin- catalytic efficiency at different substrate concen- ical severity of hemolysis in persons in different trations. In most cases the available data are inad- families (and sometimes in the same kindred) in equate for assessment of these properties. the presence of in vitro enzyme assays indicating Technological difficulties of in vitro assays like- comparably severe deficiency? What are the ul- wise exist. Since broken cells are employed, en- timate mechanisms of hemolysis in deficient ery- zyme stability in dilute hemolysates and under throcytes? preparatory conditions are factors in many in-

290 APRIL 1968 * 108 * 4 stances insufficiently explored. The glycolytic se- 5. Brok, F., Ramot, B., Zwang, E., and Danon, D.: Enzyme activities in human red blood cells of different quences are composed of interlocking and inter- age groups, Israel J. M. Sci., 2:291, 1961. dependent reactions. When a critical, rate-limiting 6. Rapaport, S., and Luebering, J.: The formation of threshold of activity for any given enzyme is ap- 2,3-diphosphoglycerate in rabbit erythrocytes: the existence of a diphosphoglycerate mutase, J. Biol. Chem., 183: proached, the phenotypic consequences are con- 507, 1950. ditioned by all the other interrelated reactions of 7. Rapaport, S., and Luebering, J.: An optical study of diphosphoglycerate mutase, J. Biol. Chem., 196:583, glycolysis. Variations within the so-called normal 1952. range, ordinarily negligible, may, in the critical 8. Bartlett, G. R.: Human red cell glycolytic intermedi- threshold zone, determine the ability or inability ates and colorimetric assay methods for free and phosphorylated glyceric acids, J. Biol. Chem., 234:449 and of erythrocytes to maintain their integrity and sur- 469, 1959. vive at any given stage in their maturation. Precise 9. Murphy, J. R.: Erythrocyte metabolism. IT. Glucose in vitro assay activities may, then, not per se cor- metabolism and pathways, J. Lab. & Clin. Med., 55:286, well with in vivo erythrocyte life span. 1960. relate 10. Beutler, E.: Glucose-6-phosphate dehydrogenase The mechanisms resulting in enzymopathies activity. In The Metabolic Basis of Inherited Disease, J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, may well be diverse. A given genic defect may editors, McGraw-Hill, New York, 2nd edition, 1960. result in the actual lack of synthesis of enzyme 11. Carson, P. E., Flanagan, C. L., Ickes, C. E., and protein or, alternatively, in the production of a Alving, A. S.: Enzymatic deficiency in primaquine-sensi- catalytically inefficient protein with or without tive erythrocytes, Science, 24:484, 1956. 12. Dacie, J. V., Mollison, P. L., Richardson, N., Sel- aberrant kinetics. In the case of some forms of wyn, J. G., and Shapiro, L.: Atypical congenital haemo- G-6-PD and PK deficiencies, it is clear that the pro- lytic anemia, Quart. J. Med., 22:79, 1953. duction of qualitatively abnormal proteins is op- 13. Selwyn, J. G., and Dacie, J. V.: Autohemolysis and other changes resulting from the incubation in vitro of red erative. In many other circumstances there is no cells from patients with congenital hemolytic anemia, valid information. The possibility of the presence Blood, 9:414, 1954. of enzyme inhibitors or absence of activators is 14. de Gruchy, G. C., Santamaria, J. N., Parsons, L. C., and Crawford, H.: Nonspherocytic congenital hemolytic also present, though less likely, since no evidence anemia, Blood, 16:1371, 1960. for these has been obtained to date. Finally, the 15. de Gruchy, G. C., Crawford, H., and Morton, D.: Atypical (nonspherocytic) congenital haemolytic anemia, existence of many instances of apparently herit- Proc. VII Cong. Internat. Soc. Hemat., Rome, 2:425, able hemolytic anemia, resembling those associated 1958 (part 1), Il Pensiero Scientifico, Rome. with known enzymopathies, but in which a point 16. Robinson, M. A., Loder, P. B., and de Gruchy, G. C.: Red cell metabolism in non-spherocytic congenital of metabolic inadequacy has not been yet ascer- haemolytic anemia, Brit. J. Haemat., 7:327, 1961. tainable, renders it certain that other defined in- 17. Valentine, W. N., Tanaka, K. R., and Miwa, S.: stances of molecular disease will emerge in future A specific erythrocyte glycolytic enzyme defect (pyruvate kinase) in three subjects with congenital non-spherocytic investigation. hemolytic anemia, Tr. A. Am. Physicians, 74:100, 1961. Note: Bibliographical references have been se- 18. Tanaka, K. R., Valentine, W. N., and Miwa, S.: to Pyruvate kinase (PK) deficiency hereditary non-sphero- lected particularly indicate historical priorities cytic hemolytic anemia, Blood, 18:784, 1961 (abstract). and comprehensive studies, and to document spe- Blood, 19:267, 1962. cific points. The vast medical literature in certain 19. Boivin, P., Piguet, H., and Galand, C.: Anomalies des enzymes de la degradation du glucose erythrocytaire, areas covered precludes complete documentation; Nouv. Rev. Franc. Hemat., 6:769, 1966. hence some significant contributions have been 20. Lausecker, C., Heidt, P., Fischer, D., Hartleyb, H., omitted. These are for the most part available in and Lohr, G. W.: Anemie hemolytique constitutionnelle avec deficit en 6-phosphogluconate-deshydrogenase, Arch. the total bibliographies of the individual references Franc. Pediat., 22:789, 1965. cited. 21. Scialom, C., Najean, Y., and Bernard, J.: Anemie RPRFTE:NCES hemolytique congenitale non-spherocytaire avec deficit incomplet en 6-phosphogluconique-deshydrogenase, Nouv. 1. Allison, A. C., and Burn, G. P.: Enzyme activity as Rev. Franc. 6:452, 1966. a function of age in the human erythrocyte, Brit. J. Hemat., Haemat., 1:291, 1955. 22. Lohr, G. W., and Waller, H. D.: Eine neue enzy- 2. Marks, P. A., Johnson, A. B., Hirschberg, E., and mopenische hamolytische Aniimie mitlGlutathion-reduk- Banks, J.: Studies on the mechanism of aging of human tase-mangel, Med. Klinik, 57:1521, 1962. red blood cells, Ann. New York Acad. Sci., 75:95, 1958. 23. Carson, P. E., Brewer, G. J., and Ickes, C.: De- 3. Marks, P. A., Johnson, A. B., and Hirschberg, E.: creased glutathione reductase with susceptibility to he- Effect of age on enzyme activity in erythrocytes. Proc. molysis, J. Lab. & Clin. Med., 58:804, 1961 (abstract). Nat. Acad. Sci., 44:529, 1958. 24. Harvald, B., Hanel, K. H., Squires, R., and Trap- 4. Sass, M. D., Vorsanger, E., and Spear, P. W.: En- Jensen, J.: Adenosine-triphosphatase deficiency in patients zyme activity as an indicator of red cell age. Clin. Chim. with nonspherocytic hemolytic anemia, Lancet, 2:18, Acta, 10:21, 1964. 1964.

CALIFORNIA MEDICINE 291 25. Necheles, T. F., Maldonado, N., Barquet-Chediak, son, H. M., Baughan, M. A., and Jaffe, E. R.: Erythro- A., and Allen, D. M.: Homozygous erythrocyte gluta- cyte enzymopathies with special reference to hemolytic thione-peroxidase deficiency, Blood, 30:880, 1967 (ab- anemia and a new specific disorder of glycolysis (phos- stract). phoglycerate kinase-PGK-deficiency). Unpublished data. 26. Oort, M., Loos, J. A., and Prins, H. K.: Hereditary 39. L8hr, G. W., and Waller, H. D.: Zur Biochemie absence of reduced glutathione in the erythrocytes-a new einiger angeborener ha.molytischer Anamien, Folia Hae- clinical and biochemical entity, Vox Sang, 6:370, 1961. mat., 8:377, 1963. 27. Prins, H. K., Oort, M., Loos, J. A., Ziircher, C., 40. Alagille, D., Fleury, J., and Odievre, M.: D6ficit and Beckers, T. H.: Hereditary absence of glutathione in congenital en 2, 3-diphosphoglyc6romutase, Bull. Mem. the erythrocytes. Biochemical, haematological and geneti- Soc. Med. Hop., Paris, 115:483, 1964. cal studies, Proc. IX Congr. Europ. Soc. Haemat., Lisbon, 41. Schr8ter, W.: Kongenitale nichtsphiarocytare Hia- 1963, p. 739 (S. Karger, Basel/New York, 1963). molytische Anamie bei 2, 3-diphosphoglycerate Mutase- 28. Prins, H. K.: Oort, M., Loos, J. A., Zurcher, C., mangel du Erythrocyten im Frohen Sauglingsalter, Klin. and Beckers, T. H.: Congenital nonspherocytic hemolytic Wschr., 43:1147, 1965. anemia associated with glutathione deficiency of the eryth- 42. Schroter, W., and Heyden, H. V.: Kinetik der rocytes. Hematologic, biochemical, and genetic studies, 2,3-diphosphoglycerateunsatzes in menschlichen Erythro- Blood, 27:145, 1966. cyten, Biochem. Zeitsch., 341:387, 1965. 29. Valentine, W. N., and Tanaka, K. R.: Pyruvate 43. Cordes, W.: Experiences with plasmochin in ma- kinase deficiency hemolytic anemia. In The Metabolic laria: preliminary reports, 15th Annual Report, United Basis of Inherited Disease, J. B. Stanbury, J. B. Wyn- Fruit Co. (Med. Dept.), 1926, p. 66. gaarden, and D. S. Fredrickson, editors, McGraw-Hill, 44. Earle, D. P., Bigelow, F. S., Zubrod, C. G., and New York, 2nd edition, 1966, p. 1051. Kane, C. A.: Studies on the chemotherapy of the human 30. Keitt, A. S.: Pyruvate kinase deficiency and related malarias. IX. Effect of pamaquine on the blood cells of disorders of red cell glycolysis, Am. J. Med., 41:762, man, J. Clin. Invest., 27:121, 1948. 1966. 45. Hockwald, R. S., Arnold, J., Clayman, C. B., and 31. Schneider, A. S., Valentine, W. N., Hattori, M., Alving, A. S.: Status of nrimaquine. IV. Toxicity of and Heins, H. L., Jr.: Hereditary hemolytic anemia with primaouine in Negroes, JAMA, 149:1568, 1952. triosephosphate isomerase deficiency, New Eng. J. Med., 46. Dern. R. J., Weinstein, I. M., LeRoy, G. V., Tal- 272:229, 1965. madge, D. W., and Alving, A. S.: The hemolytic effect 32. Valentine. W. N., Schneider, A. S., Baughan, of primaquine. I. The localization of the drug-induced M. A., Paglia, D. E., and Heins, H. L., Jr.: Hereditary hemolvtic defect in primaquine-sensitive individuals, J. hemolytic anemia with triosephosphate isomerase defi- Lab. Clin. Med., 43:303, 1954. ciency. Studies in kindreds with co-existent sickle cell 47. Dern, R. J.. Beutler, E., and Alving, A. S.: The trait and erythrocyte glucose-6-phosphate dehydrogenase hemolytic effect of primaquine. IT. The natural course of deficiency, Am. J. Med., 41:27, 1966. the hemolytic anemia and the mechanism of its self- 33. (a) Schneider, A. S., Valentine, W. N., Baughan, limited character, J. Lab. Clin. Med., 44:171, 1954. M. A., Paglia, D. E., Shore, N. A., and Heins, H. L., Jr.: 48. Beutler, E., Dern. R. J., Flanagan C. L., and (A) Triosephosphate isomerase deficiency, a multi-svstem Alving, A. S.: The hemolytic effect of primaquine. VII. inherited disorder. Clinical and genetic aspects. In Hered- Biochemical studies of drug-sensitive erythrocytes, J. Lab. itary Disorders of Erythrocyte Metabolism, E. Beutler, Clin. Med., 45:286, 1955. editor, Grune and Stratton, New York, in press, 1968. (b) Schneider, A. S., Dunn, I., Ibsen, K. H., and Wein- 49. Beutler, E.: The glutathione instability of drug- stein, I. M.: (B) Inherited triosephosphate isomerase defi- sensitive red cells: a new method for the detection of ciency. Erythrocyte carbohydrate metabolism and prelim- drug sensitivity, J. Lab. Clin. Med., 49:84, 1957. inary studies of the erythrocyte enzyme. Thid, 1968. 50. Beutler, E., Dern, R. J., and Alving, A. S.: The 34. Valentine, W. N., Oski, F. A., Paglia, D. E., hemolytic effect of primaquine. VI. An in vitro test for Baughan, M. A., Schneider, A. S., and Naiman, J. L.: sensitivity of erythrocytes to primaquine, J. Lab. Clin. Hereditary hemolytic anemia with hexokinase deficiency. Med., 45:40, 1955. Role of hexokinase in erythrocyte aging, New Eng. J. 51. Jandl, J. H., Engle, L. K., and Allen. D. W.: Oxi- Med., 276:1, 1967. dative hemolysis and precipitation of hemoglobin, J. Clin. 35. Valentine, W. N., Oski, F. A., Paglia, D. E., Invest., 39:1818, 1960. Ibid., 40:454, 1961. Baughan, M. A., Schneider, A. S., and Naiman, J. L.: 52. Szeinberg, A., Sheba, C., Hirshorn, N., and Bodo- Erythrocyte hexokinase and hereditary hemolytic anemia. nonyi, E.: Studies on erythrocytes in cases with a past In Hereditary Disorders of Erythrocyte Metabolism, E. history of favism and drug-induced acute hemolytic Beutler, editor, Grune and Stratton, New York, in press, anemia, Blood, 12:603, 1957. 1968. 53. Szeinberg, A., Asher, Y., and Sheba, C.: Studies 36. Baughan, M. A., Valentine, W. N., Paglia, D. E., on glutathione stability in erythrocytes of cases with past Ways, P. O., Simon, E. R., and DeMarsh, Q. B.: Heredi- history of favism or sulfa-drug-induced hemolysis, Blood, tary hemolytic anemia associated with glucosephosphate 13:348, 1958. isomerase (opi) deficiency -a new enzyme defect of 54. Szeinberg, A., Sheba, C., and Adam, A.: Enzymatic human erythrocytes, Amer. Society of Hematology Meet- abnormality in erythrocytes of a population sensitive to ing, Toronto, Canada, Dec. 1967, Blood, 30:850, 1967 Vicia faba or drug-induced hemolytic anemia, Nature, (abstract). London, 181:1256, 1958. 37. Baughan, M. A., Valentine. W. N., Paglia, D. E., 55. Zinkham, W. H., Lenhard, R. E., Jr., and Childs, Ways, P. O., Simon, E. R., and DeMarsh, Q. B.: Heredi- E.: A deficiency of glucose-6-phosphate dehydrogenase tary hemolytic anemia associated with glucosephosphate from with favism, Bull. Johns isomerase (GPI) deficiency - a new enzyme defect of in erythrocytes patients human erythrocytes, Blood, in press. Hopkins Hosp., 102:169, 1958. 38. (a) Kraus, A. P., Langston, M. F., and Lynch, 56. Sansone, G., Piga, A. M., and Segni, G.: I1 favisma, B. L.: Red cell phosphoglycerate kinase deficiency. A new E-dizioni Minerva Medica, Torino, 1958. cause of nonspherocytic hemolytic anemia, Biochem. 57. Szeinberg, A., Sheba, C., and Adam, A.: Selective Biophys. Res. Comm., 30:173, 1968. occurrence of glutathione instability in red blood corpus- (b) Valentine, W. N., Hsieh, H., Paglia, D. E., Ander- cles of the various Jewish tribes, Blood, 13:1043, 1958.

292 APRIL 1968 * 108 * 4 58. Newton, W. A., Jr., and Bass, J. C.: Glutathione- mit hiimatologischen und neurologischen Storungen, Klin. sensitive chronic nonspherocytic hemolytic anemia, Amer. Wchnschr., 43:413, 1965. J. Dis. Child., 96:501, 1958. 78. Mills, G. C.: Hemoglobin catabolism. L. Gluta- 59. Shahide, N. T., and Diamond, L. K.: Enzyme defi- thione peroxidase, an erythrocyte enzyme which protects ciency in erythrocytes in congenital nonspherocytic hemo- hemoglobin from oxidative breakdown, J. Biol. Chem., lytic anemia, Pediatrics, 24:245, 1959. 229:189, 1957. 60. Kirkman, H. N., and Riley, H. D., Jr.: Congenital 79. Mills, G. C., and Randall, H. P.: Hemoglobin nonspherocytic hemolytic anemia, Am. J. Dis. Child., catabolism. II. The protection of hemoglobin from oxi- 102:313, 1961. dative breakdown in the intact erythrocyte, J. Biol. 61. Motulsky, A. G., and Campbell-Krant, J. M.: Chem., 232:589, 1958. Population genetics of glucose-6-phosphate dehydrogenase 80. Necheles, T. F., Boles, T. A., and Allen, D. M.: deficiency of the red cell, Proc. Conf. Genetic Poly- Erythrocyte glutathione-peroxidase deficiency and hemo- morphisms and Geographic Variations in Disease, Grune lytic disease of the newborn, Blood, 28:988, 1966 (ab- and Stratton, New York, 1961, p. 159. tract). 62. Allison, A. C., and Clyde, D. F.: Malaria in Afri- 81. (a) Nathan, D. G., Oski, F. A., Miller, D. R., and can children with deficient erythrocyte glucose-6-phos- Gardner, F. H.: Life span and organ sequestration of red phate dehydrogenase, Brit. M. J., 1:1346, 1961. cells in pyruvate kinase deficiency, N. Eng. J. Med., 278: 63. Kirkman, H. N., and Hendrickson, E. M.: Sex- 73, 1968. linked electrophoretic difference in glucose-6-phosphate (b) Bowman, H. S., and Procopio, F.: Hereditary non- dehydrogenase, Am. J. Human Genet., 15:241, 1963. spherocytic hemolytic anemia of pyruvate kinase deficient 64. Boyer, S. H., Porter, I. H., and Weilbacher, R. G.: type, Ann. Int. Med., 58:567, 1963. 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CALIFORNIA MEDICINE 293 95. Waller, H. D., and Lohr, G. W.: Hereditary non- 102. Busch, D., Witt, I., Berger, M., and Kunzer, W.: spherocytic enzymopenic hemolytic anemia with pyruvate Deficiency of pyruvate kinase in the erythrocytes of a kinase deficiency, Proc. IX Cong. Internat. Soc. Hemat., child with hereditary non-spherocytic hemolytic anemia, Mexico City, 1962, Universidad Nacional Autonoma de Acta Paed. Scand., 55:117, 1966. Mexico, Mexico, D.F., p. 257. 103. Sachs, J. R., Wicker, D. J., Gilcher, R. O., Conrad, 96. Rose, I. A., and Warms, J. V. B.: Control of glycol- M. E., and Cohen, R. J.: PK deficient hemolytic anemia ysis in the human red blood cell, J. Biol. Chem., 241: inherited as an autosomal dominant, American Society of 4848, 1966. Hematology Meeting, Toronto, Canada, Dec. 5, 1967, 97. Twomey, J., Moser, R., Hudson, W., O'Neal, F., Blood, 30:881, 1967 (abstract). and Alfrey, C.: ATP metabolism in pyruvate kinase (PK) 104. Shore, N. A., Schneider, A. S., and Valentine, deficient erythrocytes, Clin. Res., 14:437, 1966 (abstract). W. N.: Erythrocyte triosephosphate isomerase deficiency, J. Pediat., 67:939, 1965. 98. Hsu, T. H. J., Robinson, A. R., and Zuelzer, W. W.: 105. Valentine, W. N., Oski, F. A., Paglia, D. E., Genetic polymorphism of hemolytic anemias associated Baughan, M. A., Schneider, A. S., and Naiman, J. L.: with erythrocyte pyruvic kinase deficiency, Blood, 28: Erythrocyte hexokinase and hereditary hemolytic anemia. 977, 1966 (abstract). In Hereditary Disorders of Erythrocyte Metabolism, E. 99. Boivin, P., and Galand, C.: Constante de Mi- Beutler, editor, Grune and Stratton, New York, in press, chaelis anormale pour le phospho-enol-pyruvate au cours 1968. d'un deficit en pyruvate-kinase erythrocytaire, Rev. Franc. 106. Paglia, D. E., Holland, P., Valentine, W. N., and etudes clin. et biol., 12:372, 1967. Baughan, M. A.: Unpublished data. 100. Miwa, S.: Personal communication. 107. Detter, J. C., Ways, P. O., Giblett, E. R., Baughan, 101. Paglia, D. E., Valentine, W. N., Baughan, M. A., M. A., Hopkinson, D. A., Povey, S., and Harris, H.: In- Miller, D. R., Reed, C. F., and McIntyre, 0. R.: Identifi- herited variations in human phosphohexose isomerase, cation of an isozyme of pyruvate kinase (PK) responsible Ann. Human Genet., in press, 1968. for hereditary hemolytic anemia, American Society of 108. Bowdler, A. J., and Prankerd, T. A. J.: Studies in Hematology Meeting, Toronto, Canada, Dec. 5, 1967, congenital non-spherocytic hemolytic anemias with spe- Blood, 30:881, 1967 (abstract). cific enzyme defects, Acta Haemat., 31:65, 1964.

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