Metabolic abnormalities of erythrocytes from patients with congenital nonspherocytic hemolytic anemia

METABOLIC ABNORMALITIES OF ERYTHROCYTES FROM WITH CONGENITAL NONSPHEROCYTIC HEMOLYTIC WILLIA~

I-I.ZINK~IAM, M.D., A~WD R. E.

PATIENTS ANEMIA

LENHARD, JR., M.D.

BALTIMORE, MD. | N RECENT years th e mechanism of I hemolysis in a variety of hemolytic disorders has been p a r t i a l l y elucidated through studies on the biochemical characteristics of normal as well as abnormal erythrocytes. A noteworthy example of the fruitfulness of such an approach was the demonstration by Alving and his associates of the altered metabolism in red cells from individuals susceptible to the hemolytic effects of certain drugs and chemicals : primaquine, phenacetin, sulfanilamide, naphthalene, Furadantin, Gantrisin, Kynex, and others. This unique sensitivity to hemolysis was related to an " i n b o r n error of red cell metabolism" which apparently is innocuous until persons with these erythrocytes are exposed to potentially hemolytic compounds. A comprehensive review of the role of various investigators in elucidating the mechanism and genetic transmission of drug-induced hemolysis was recently published by Beutler. 1 Drugsensitive erythroeytes are not detectable by routine hematologic procedures such as examination of smears of the peripheral blood, the Coombs test, electrophoretic analysis of hemoglobin, and osmotic and mechanical F r o m t h e D e p a r t m e n t of P e d i a t r i c s , T h e J o h n s H o p k i n s School of [Yledicine, a n d t h e Harriet Lane Home, The Johns Hopkins Hospitals. T h i s i n v e s t i g a t i o n w a s s u p p o r t e d b y a res e a r c h grant, H-3995, f r o m the N a t i o n a l Ins t i t u t e s of H e a l t h , P u b l i c H e a l t h S e r v i c e .

fragility tests. The description of certain biochemical alterations in these red cells, however, has resulted in the development of relatively simple in vitro tests for their detection. Drugsensitive erythrocytes have a slight deficiency of reduced glutathione (GSH) as well as a marked fall in GSH when whole blood is incubated with acetylphenylhydrazine (GStt stability test). Another abnormality is a reduction of glucose-6-phosphate dehydrogenase activity, the enzyme which initiates the hexose monophosphate shunt. As was predicted, these biochemical alterations are genetically determined and are probably transmitted by a sex-linked gene with variable expressivity. The racial distribution and intraraciM frequency of these red cell defects have also been studied. Drug-sensitive erythrocytes have been described in American Negroes, Orientals, Greeks, Italians, and Sephardie Jews. In the first group approximately 15 per cent of the men and 2 per cent of the women manifest a marked deficiency of red cell glucose-6-phosphate dehydrogenase activity, while 5 per cent of the women have only a moderate decrease of enzyme activity. Men and women with a marked reduction of glucose-6-phos9 phate dehydrogenase activity have been designated " r e a c t o r s , " whereas those women with a partial decrease 319

320

T H E J O U R N A L OF PEDIATRICS

in red cell enzyme activity have been termed "intermediates." Intermediate degrees of the enzyme deficiency have not been found in Negro men. The term " c o n g e n i t a l nonspherocytic hemolytic a n e m i a " has been applied to congenital and sometimes familial hemolytic disorders which are not associated with a characteristic alteration of red cell morphology. 2s The diagnosis is made, therefore, only after congenital hemolytic diseases identifiable by abnormally shaped e r y t h r o c y t e s have been excluded (Mediterranean anemia, h e r e d i t a r y spherocytosis, the hemoglobinopathies, and h e r e d i t a r y elliptocytosis). A review of the case reports of patients with " c o n g e n i t a l nonspherocytic hemolytic a n e m i a " reveals eertain features which are common to all of them: the osmotic and mechanical fragility studies are either normal or only slightly increased; the life span of the patient's e r y t h r o e y t e s in an autologous or homologous environment is r e d u c e d ; splenectomy is usually not beneficial; the disease appears to be transmitted as a Mendelian dominant. Other findings, however, indicate t h a t not all of these patients have the same hemolytic disorder. In some the red ceils are heavily stippled 6, 7 and in others the rate of antohemolysis in vitro is greatly aecelerated. 6, ' Hence, it seems likely t h a t the term "congenital nonspherocytic hemolytic a n e m i a " as it is c u r r e n t l y used signifies a polyglot group of hemolytic conditions. V e r y little is k n o w n about the nature of the biochemical defect which shortens the survival time of nonspherocytic erythrocytes. B y studying the effect of glucose on autohemol-

ysis, on the increase in osmotic fragility, and on cation changes of red ceils incubated in vitro, Selwyn and Daeie 9 were able to distinguish two varieties of " c o n g e n i t a l nonspherocytic hemolytic a n e m i a . " More recently Newton and Bass 1~ r e p o r t e d an abnormality of g]utathione metabolism and a total absence of glucose-6phosphate dehydrogenase activity in red cells from three Italian chiIdren with nonspherocytic hemolytic anemia. Similar biochemical abnormalities were not demons{raMe in the parents of these patients. During the past 2 years we have observed 6 cases of congenital nonspherocytic hemolytic anemia from 5 unrelated families. One of these patients, a Caucasian boy, developed hemoglobinuria following ingestion of Phenacetin, and biochemical abnormalities comparable to those described in prim.aquine-sensitive individuals were demonstrated in his erythrocytes. Following this observation, similar studies were done on the other 5 patients, and in the present communication the results and significance of these findings are presented and discussed. CASE REPORTS

CASE 1 . - - P a t i e n t E. R., a 6-year-old white boy, was r e f e r r e d to this hospital for investigation of a chronic hemolytic anemia. He has observed to be icteric 24 hours after delivery, but no AB-O or R H incompatibility was demonstrated. The jaundice disappeared in one week, and the patient was considered normal until 2 years of age. Because of pallor, his peripheral blood was examined, and the parents were told .that he had an unusual type of hemolytic anemia. The patient remained asymptomatic, and periodic blood counts revealed that

D.

C.

Fig. 1.--Smears o f p e r i p h e r a l b l o o d s t a i n e d w i t h ~VVrigbt's s t a i n ( X 7 5 0 ) . A, B l o o d f r o m C a s e 1 o n F e b . 12, 1958. The red cells are n0rmocytic and an occasional macrocyte is p r e s e n t . B, B l o o d f r o m t h e m a t e r n a l g r a n d f a t h e r o f C a s e s 1 a n d 2 o n A p r i l 3, 1958. T h e r e d c e l l s a r e n o r m a l . C, B l o o d f r o m C a s e 3 o n N o v . 24, 1958. The red cells are D, ]Blood f r o m C a s e 4 o n S e p t . 13, 1956. T h e r e a r e m a e r o c y t o s i s a n d s l i g ' h t h y p o e h r o m i a a n d s o m e o f t h e c e l l s normal. are target shaped. T h e n u c l e a t e d red b l o o d cell is a n o r m o b i a s t ( r e d u c t i o n 1~).

B.

A.

bD

O

.o

SUBJECT

14.5 22.0 23.9

17.6

30.0 30.5

47.0 38.9 41.6

4.1 5.9 6.1

6.1

5.0

9.3 8.4

15.0 12.4 13.4

5/18/41

7/27/54 7/14/58 Family S Father Mother Case 6

16,800 32,500 13,750 6,900 38~000 8,500 9,650

8,800

10.0 12.0 24.0 43.0

1.2 1.6 0.5

4,300

6,100

7,000

6,000

8,700

5,750

4,850

5,750

(UNCORRECTED)

W K I T E BLOOD CELL .COUNT

14.0 37.8 46.2

10.8

1.6

37.7

11.8

33.5

2.4

37.5

11.4

11.2

1.4

46.0

14.3

2.5

7.6

0.8

10.0

1.6

2.5

(%)

RETICULOCYTE COUNT

HEREDITARY

0

0

2

2 17

81 10

12

0

0

0

0

0

0

0

0

0

0

0

CELLS

Normal Normal Normal

150,000 444,000 Normal 746,000

252,000 660,000 Increased

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

PLATELETS

0.6

5.0

3.5 0.4

1.2 1.0

2.0

1.2

0.6

1.0

1.2

1.4

0.74

0.58

[

0.2

<0.8

0.56 0.1

<0.8 <0.8

0.2

0.2

0.2

0.1

0.2

0.1

0.11

0.12

DIRECT

(MG. %)

BILIRUBIN

HEMOLYTIC

TOTAL

NONSPHEROCYTIC

I NUCLEATED RED I ]BLOOD CELLS PER I 100 WHITE BLOOD I

D A T A ON PATIENTS W I T H

15.0

35.0

42.5

Case 4 7/23/48 8/5/49 9/13/56 Case 5 9/13/40

11/29/58

Case 3

:Family K Father 11/24/58 Mother 11/24/58 Brother 11/24/58

4/3/58

Maternal grandfather

4/3/58

10.8

37.0

~I12158

32.0

34.0

Case 2 2/12/58 Maternal grandmother

40.0

47.0

(%)

2/12/58

13.0

(aM. %)

HEMATO CRIT

Case 1 2/12/58 Brother

Mother

2/12/58

Family R Father

HEMOGLOBIN

T A B L E I. HEMATOLOGIC

Negative

Negative

Negative

Negative

Negative

Negative

DIRECT COOMBS TEST

ANEI~IA

Oct., 1947

11/1/40

3/7/49

(DATE OF)

SPLENECTOMY

C~ 5rk

O e~

Z

9

['3 s

ZINKHAM AND LENtIARD:

HEMOLYTIC ANEMIA

323

the hemoglobin r a n g e d between 10 and 12 Gm. per cent and the reticulocyte count between 6 and 10 per cent. At 53/~ years of age he developed an u p p e r r e s p i r a t o r y infection which was t r e a t e d with one injection of Bicillin and 3 " f e v e r t a b l e t s , " each containing 2.5 Gr. of Phenacetin. One day later the patient was v e r y pale, his sclerae were icteric, and he voided black urine. He was admitted to another hospital where the following l a b o r a t o r y d a t a was learned: hemoglobin was 7.4 Girt. per cent; hematocrit, 27.0 per cent; white blood cell count, 5,800 per cubic millimeter; reticulocyte count, 4.7 per cent; direct Coombs test, negative; serologic studies for cold and acid hemolysins were negative; electrophoresis of hemoglobin, AA p a t t e r n ; total bilirubin, 0.58 rag. per cent (direct was 0.12 rag. per cent) ; blood urea nitrogen, 7.2 rag. per cent; nonincubated osmotic fragility studies were normal. No intact red cells were seen in the urine, but the test for occult blood was positive. The color of the urine r e t u r n e d to normal over the n e x t 2 days, and the patient was discharged on the t e n t h hospital d a y with a hemoglobin value of 10.6 Gin. per cent. Five weeks later the patient was evaluated at this hospital. The sclerae were white, the h e a r t was not enlarged, and the liver and spleen were not palpable. Hematologic data are listed in Table I, and morphology of the e r y t h r o c y t e s is illustrated in Fig. I.

reticulocyte values remained unchanged, and he continued to do well until 2 years of age. At this time an u p p e r r e s p i r a t o r y infection occurred which was t r e a t e d with tetracycline, and although the f e v e r and cough subsided, m a r k e d pallor ensued and he was hospitalized. The physical examination on admission was normal except for moderate pallor of the skin. The sclerae were not icteric and the liver and spleen were not palpable. The following l a b o r a t o r y Studies were obtained: hematocrit was 27.0 per cent; reticuloeyte count, 20.0 per cent; total white blood cell count, 20,700 per cubic millimeter; a differential count of the leukocytes showed 27 per cent polymorphonuclear neutrophils, 65 per cent lymphocytes, 7 per cent eosinophils, and 1 per cent basophils; direct Coombs test negative; paper eleetrophoresis of hemoglobin, hemoglobin AA; total serum bilirubin, 0.74 rag. per cent (direct was 0.11 mg. per cent); nonincubated osmotic fragility study, normal; urinalysis, normal. No specific t h e r a p y was given to the patient and he was discharged on the fifth hospital day. W h e n seen at this hospital 6 weeks later, the physical examination was normal. The hematologic data obtained on that visit are r e p o r t e d in Table I, and the morphology o f : t h e e r y t h r o e y t e s is described in Fig. 1. No Heinz bodies were seen in the red cells and the nonincubated and incubated osmotic fragilities were normal.

CASE 2.--Ed. R., the brother of E. R. (Case 1), is 21~ years old and has been followed at another hospital because of a hemolytic anemia first observed at i y e a r of age. The m o t h e r ' s pregn a n c y with this patient was normal and the patient had no clinical icterus during the neonatal period. A t 11 months of age he was noted to be pale, and an examination of the peripheral blood several weeks later revealed a hemoglobin of 10 Gin. per cent and a reticulocyte count of 15 per cent. Subsequently, the patient's clinical condition and hemoglobin and

Family History (Cases E. R. and Ed. R . ) . ~ A partial pedigree of this family is outlined in Fig. 2. Both parents and the brother of these patients are living and well and no abnormalities were demonstrable in their peripheral blood. The maternal grandf a t h e r is 56 years old, and he has experienced episodes of jaundice since childhood which are usually assoMated with the passage of d a r k yellow urine. A t 36 years of age extensive blood studies were done at another clinic, and following these the

"4 . ,~2

THE

J O U R N A L OF :PEDIATRICS

p a t i e n t was told t h a t [he h a d a " c o n genital n o n s p h e r o e y t i e hemolytic anem i a . " A e h o l e e y s t e e t o m y was done at 40 y e a r s of age because of gallstones. Since t h a t time the patient has not been a w a r e of ieterus, although there h a v e been occasional episodes of

ond bout of jaundice was told t h a t he had a hemolytic anemia. His general health since t h a t time, however, has been excellent. The m e m b e r s of this f a m i l y are A m e r i c a n with m i x e d English, French, and G e r m a n i c antecedents. FAMIL.Y

FAMILY

"R"

"K ~

!

CASE 3 CASE I NORMAL ]BLOOD

G - 6 -P.D.

MARI~ED

PERIPHERAL COUNT

]

AND

ACTIVITY

HEMOLYTIC ANEMIA NBY H I S T O R Y . G-6-P.D. S T U D I E S N O T DONE 2.--l~amily

pedigree

CASE 2

DEFICIENCY

OF G - 6 - P , D . MODERATE DEFICIENCY OF G - 6 - P . D . AND~OR INTERMEDIATE GSH STABILITY TEST

HEMOLYTIC ANEMIA ]]]]] VE R IF IE D BY PERIPHERAL BLOOD COUNTS

Fig.

J-~

N

of Cases

fatigue and dyspnea. The most recent p e r i p h e r a l blood findings in this m a n are listed in Table I. There are 3 m a t e r n a l greatuneles: one died at 40 y e a r s of age and the cause of d e a t h is u n k n o w n ; the second died at 60 y e a r s of age while being t r e a t e d f o r a hemolytic a n e m i a ; the t h i r d is 68 y e a r s old and is occasionally transfused because of a chronic hemolytic anemia. The ancestral b a c k g r o u n d of this family is Germanic and English, and the family pedigree has been t r a c e d t h r o u g h four generations. CASE 3.--This 10-year-old white boy, patient A. K., was seen because of intermittent episodes of jaundice and anemia since birth. Both parents and an 8-year-old b r o t h e r are living and well. A m a t e r n a l uncle, 40 y e a r s of age, had t r a n s i e n t ieterus at the age of 9 and 18 years and d u r i n g the see-

DEAD. NO HISTORY OF HEMOLYTIC ANEMIA

i and

2 and

Case

3.

The patient was first noted to be j a u n d i c e d at 2 m o n t h s of age. The parents were told that their child was slightly anemic and an oral iron preparation was prescribed. The patient was asymptomatic until 5 years of age

w h e n jaundice r e c u r r e d and persisted for a period of 3 months. Since t h a t time there have been three episodes of jaundice, all preceded b y an up'per r e s p i r a t o r y infection and all assoMated with severe anemia and passage of d a r k orange-colored urine. D u r i n g two of these a t t a c k s he was hospitalized and t r a n s f u s e d with whole blood. The p a t i e n t has received short courses of penicillin, tetracycline, A u r e o m y cin, and acetylsalieylie acid, but the onset of the ieterie-anemia spells was n o t r e l a t e d to the a d m i n i s t r a t i o n of these drugs. W h e n seen at this hospital the patient was a well-developed and wellnourished boy. The sclerae were

ZINKHAM

AND LENHARD:

white and the liver and spleen were not palpable. 'Hematologic d a t a on the patient and his family are recorded in Table I ; the appearance of the patient's e r y t h r o c y t e s is illust r a t e d in. Fig. 1. Nonincubated and incubated osmotic and mechanical fragility studies were n o r m a l CASE 4 . - - P a t i e n t R. J. is a 12-yearold American Negro girl who was t r a n s f e r r e d to this hospital at 10 days

HEMOLYTIC

325

ANEMIA

and the patient was transferred to this hospital. On admission the patient was pale, listless, and intensely jaundiced. The edges of the liver and spleen were p a l pable 3 era. below the costal margin. Routine l a b o r a t o r y studies revealed: hemoglobin was 4.2 Gin. per cent; the total nucleated cell count was 11,000 per cubic millimeter; the differential count showed 50 per cent polymorphonuclear neutrophi]s, 42 per cent

t~

< r (..9 z

1 5 84

IO

,,,n 0 ,,J L9 0

5

bJ

"r u') uJ

>-

o I---

0

bJ

z bJ .J

>" U O "J _U Fo~ D 401 20

0 U~

5O

r I-

u

ill IILI III I : I

I

2

AGE Fig.

IN

I

I

I

I

3

4

5

6

xl 7

I

I

I

8

9

I0

YE ARS

3 . - - V a r i a t i o n s of h e m o g l o b i n a n d r e t i c u l o c y t e v a l u e s a n d f r e q u e n . e y of t r a n s f u s i o n s C a s e 4, S p l e n e c t o m y w a s p e r f o r m e d s~t 1 10/12 y e a r s of a g e .

of age because of anemia and jaundice. The patient's birth weight was 5 pounds, 9 ounces, and the perinatal course was u n e v e n t f u l until the fifth day of life. A~ this time skin and scleral icterus appeared, the edge of the spleen was felt 4 cm. below the costal margin, and the hemoglobin was 5 Gin. per cent. A transfusion os 18 ml. of whole blood was given into the superior sagittal sinus. Following this procedure the icterus deepened

in

lymphocytes, 7 per cent monocytes, with 8 nucleated e r y t h r o c y t e s per 100 leukocytes; three blood cultures were sterile; urinalysis was normal; the total bilirubin was 16.8 rag. per cent (direct, 1.2 rag. per c e n t ) ; the serologic test for syphilis and the direct Combs test were negative; the patient's blood group was 0 M N Rhj ; no circulating Rh antibodies were demonstrable in the m o t h e r ' s serum. P a p e r electrophoresis of hemoglobin

326

THE

JOURNAL

done when the patient was 10 years of age revealed only hemoglobin A. The nonincubated and incubated osmotic and mechanical f r a g i l i t y tests were each normal when the patient was 8 and 10 years of age, respectively. As shown in Fig. 3 the patient was r e g u l a r l y transfused e v e r y 4 to 6 weeks d u r i n g the first 2~/~ years of her life. Following splenectomy at 3 years of age the recticu]ocyte count rose to a range of 20 to 40 per cent, the hemoglobin stabilized at a level of 8 Gin. per cent, and only two transfusions have been given in a period of 9 years since the operation. Detailed hematologic data before and a f t e r splenectomy are listed in Table I, and m o r p h o l o g y of the e r y t h r o c y t e s is depicted in Fig. 1. The parents, American Negroes, are living and well and their peripheral blood findings are normal. CASE 5 . - - P a t i e n t R. C., a 19-year-old white boy, was first seen at 11~ years of age, with a history of jaundice and r e c u r r e n t anemia from 2 days of age. The parents, two brothers, and one sister are living and well. t~eripheral blood findings in the parents are normal. The m o t h e r ' s p r e g n a n c y with this patient and the delivery were normal. Icterus of the skin and sclerae was observed at 2 days of age. The intensity of the jaundice lessened and t h e baby was well until 7 weeks of age. During an u p p e r r e s p i r a t o r y infection m a r k e d pallor was observed and he was transfused with whole blood. Two subsequent transfusions were given at 3 and 16 months of age, and the patient was r e f e r r e d to this hospital for f u r t h e r studies at 18 months of age. At this time the skin and conjunctivae were pale, the selerae were slightly icterie, the frontal and parietal bones of the skull were prominent, and the edges of the liver and spleen were felt 3 cm. below the costal margin. Admission laborat o r y findings r e v e a l e d : hemoglobin was 5.4 Gin. per cent; the leukocyte count was 8,600; the differential count

OF PEDIATRICS

showed 37 per cent polymorphonuclear neutrophils, 6 per cent juvenile neutrophils, 49 per cent lymphoeytes, 5 per cent monoeytes, 2 per cent eosinophils, and 1 per cent basophils. There were 2 nucleated erythroeytes per 100 leukocytes, and the retieuloeyte count was 10 per cent. Urinalysis was normal. The total bilirubin was 3.7 mg. per cent (direct, 0.9 rag. per cent). While in the hospital the patient developed an u p p e r respirat o r y infection, and a pneumoeoccus, type VI, was cultured from the nasop h a r y n x ; sulfapyridine was given by mouth for a period of 10 days. One week later the patient was given two transfusions of whole blood and 24 hours later a splenectomy was performed. At the time of discharge, 3 weeks later, the hemoglobin was 10.1 Gm. per cent and the reticuloeyte count was 3.5 per cent. Since splenect o m y the patient has received only one transfusion. Slight pallor and sclera] ieterus have persisted, and the color of the urine becomes d a r k yellow during infections. Detailed hematologic data obtained before and after splenectomy are recorded in Table I. A total of 5 osmotic fragility studies, 2 done on nonincubated and incubated whole blood, were normal. Two nonincubated and incubated mechanical fragility tests were normal. The appearance of the e r y t h r o c y t e s in smears of the peripheral blood is shown in Fig. 1. CASE 6.--This 15-year-old white girl has been extensively studied by L a r g e and Akeroyd 11 and was recently reported as a case of " c o n g e n i t a l inclusion body hemolytic anemia with abnormal pigment metabolism." In r6sum6, patient " K . S . " has had a hemolytic anemia associated with the passage of dark urine since 21/~ years of age. She required transfusions of whole blood every 2 to 3 months until the age of 10 years, after which time no f u r t h e r transfusions were necessary. The father's blood was used for many of these transfusions since the administration of serologic compatible blood

ZINKHAM

AND

LENHARD:

froln other donors was sometimes associated with severe hemolytic reactions. Other noteworthy features of this ease are the r e c u r r e n t u p p e r respiratory infections, the occurrence of which has been unaffected b y two tonsillectomies and adenoidectomies; the persistent petit mal and g r a n d nml seizures which have been less frequent since the menarehe at 11 years of age; the presence of inclusion bodies in a p p r o x i m a t e l y 14 p e r cent of the red blood ceils; fasting blood sugars which have ranged between 27.8 and 54.0 rag. per cent. The patient has received Mesantoin, Tridione, Dilantin, and phenobarbital for her epileptic seizures and at present she is reeeiving Dilantin. Detailed hematologic data as welt as a description of the inclusion bodies of the erythroeytes, red cell survival studies, in vitro tests for Heinz body formation with phenylhydrazine, and characterization of the u r i n a r y pigment are reported in the article b y Lunge and Akeroyd2 ~ The current peripheral blood findings in the patient and her parents are listed in Table I. The ancestry of this family is English, Irish, and American Indian and there is no historical evidence of a hemolytic anemia in other members of the family pedigree. METIIODS ~

Routine hematologic techniques as well as osmotic and mechanical fragility studies were p e r f o r m e d according to methods described by H a m Y Blood for these p r o c e d u r e s was obtained either b y finger or venipuneture. The amount of reduced glutathione (GSH) in whole blood was d e t e r m i n e d *For the abbreviations

sake are

of brevity, the following used in the text:

API=I = aeetylphenylhydrazine, G-6-P ~ glucose-6-phosphate, G - 6 - P . D . ~ glucose-6-phospha.te dehydrogenase, GSI-I --~ reduced glutathione, 6-PG ---- 6-Dhosphogluconate, I~U-5-P --- ribulose-5-!ohosphate, TPN ---- triphospho!oyridine nucleotide, TPN~ ~ reducect triphosphopyridine nucleotide.

HEMOLYTIC

ANEMIA

327

b y the method of G r u n e r t and Phillips as modified by Beutler and assoeiatesY Values of G S H in venous blood obtained from 130 n o r m a l white adult men and women r a n g e d between 53 and 84 rag. per 100 ml. of erythrocytes. The stability of G S H in red blood cells was determined by the method of Beutler. 14 Whole blood, 1.0 ml., was incubated with 5.0 rag. of acetylphen ylhydrazine ( A P K ) for 2 hours at 37 ~ C., and the concentration of GSH was assayed before and a f t e r the period of incubation. In some of the determinations suffieient g h e o s e was added to whole blood to give a final concentration of added glucose of 200 rag. p e r cent. Three characteristic p a t t e r n s of response h a v e been described for this method. I n normal adults the postincubation concentration of G S H in e r y t h r o c y t e s is g r e a t e r t h a n 40 rag. per 100 ml. of red blood cells; in a p p r o x i m a t e l y 5 per cent of Negro females the values v a r y between 22 and 40 rag. per 100 m]. of e r y t h r o e y t e s ; in 14 per cent of Negro males a n d 2 p e r cent of Negro females levels of G S H are less t h a n 20 rag. per 100 ml. of red blood cells25 Those individuals w i t h values of G S H between 22 and 40 mg. per 100 m]. of erythroeytes h a v e been designated " i n t e r m e diate r e a c t o r s , " whereas persons with concentrations of G S I I less t h a n 20 mg. p e r 100 ml. of red blood cells have been t e r m e d "reactors." On the basis of f a m i l y studies now in progress it has been d e t e r m i n e d t h a t some of the "intermediate r e a c t o r s " have postincubation G S H values as high as 45 nag. p e r 100 ml. of e r y t h r o e y t e s so t h a t t h e r e is an overlap in the values of n o r m a l and " i n t e r m e d i a t e t e a t tots,

'

328

T~IE

JOURNAL

OF PEDIATRICS

A c t i v i t y of glucose-6-phosphate deh y d r o g e n a s e (G-6-P.D.) in the erythroeyte was d e t e r m i n e d b y two methods. The first of these was a modification of one r e p o r t e d b y G]aser and

an optical density change at 340 m~ of 2.07 per minute per 3.0 ml. of reaction mixture, an optical density change which represents the reduction of I / z m of TPN.

(1) G-6-P + T P N § g]ucose-6-phosphate 6-phosphogluconolactone + T P N H + H + dehydrogenase (2) 6-phosphogluconolactone

lactonase 6-PG

(6-phosphogluconate)

(3) 6-PG + T P N + 6-phosphogluconic RU-5-P (ribulose-5-phosphate) + C02 + dehydrogenase TPNH + II+ Sum of (1), (2), and (3) : G-6-P + 2 T P N § . . . . . . . . . . . .

RU-5-P + C02 + 2 T P N H + 2 H +

B r o w n 16 and has been described in a previous communicationS7; the second was p a t t e r n e d a f t e r the m e t h o d of Glock and lVfcLean2 s I n the first m e t h o d the change in optical density at 340 m~ in a B e c k m a n model DU s p e c t r o p h o t o m e t e r is followed e v e r y TABLE

II.

~OMPARISON

GI~ ~kCTIVITIES

The rationale for the second method is based on the following observations. F o r each mole of G-6-P oxidized during the first three steps of the hexose m o n o p h o s p h a t e shunt, 2 moles of reduced t r i p h o s p h o p y r i d i n e nucleotide ( T P N H ) can be formed. GF GLUCGSE-62PHGSPIIATE

I)EIIYDRGGENASE

GLUCOSE-6-PHOSPHATEDEIIYDRGGENASEACTIVITY (u./1OO ML. G r E R Y T ~ G C Y T E N ) GLUCOSE-6-PHGSP~ATE AS SUBSTRATE SUBJECTS

Normal white a d u l t men a n d wo men N e g r o women intermediate reactors Men r e a c t o r s Greek Sephardic Jew Amerlcaa Negro N e g r o women reactors These

activities

are

NUMBER

RANGE

2]

~ D E ~ I V E D 7~

AVERAGE

RANGE

180-278

219

135 186

155

13

91-198

159

59-193

82

1 ] 5

6 l 6-56

0 0 2-2,t

13

5 determined

I

30

22-95 by two

62

different

assay

techniques

1

6-44 in erythroeytes

AVERAGE

27 from

nor-

real w h i t e adults, N e g r o women " i n t e r m e d i a t e reactors," a n d men a n d w ome n " r e a c t o r s " of three

ra~ial

groups.

minute between 5 and 15 minutes a f t e r the addition of t r i p h o s p h o p y r i dine nucleotide ( T P N ) to a m i x t u r e of glueose-6-phosphate (G-6-P) and hemo]ysate. One unit of enzyme is defined as t h a t a m o u n t which produces

All of the enzymes required for these reactions are present in normal hemolysates. Therefore, a n y assay technique f o r G-6-P.D. a c t i v i t y using G-6-P as substrate and quantified by rate of T P N H formation m a y give falsely

ZINKHA5[

AND LENHARD:

high values. In order to measure only G-6-P.D. activity Glock and McLean ~8 have devised the following method. Rate of T P N H formation is measured at p t I 8.0 in one cuvette with 6-PG as substrate (0.1 ml. of a 0.02 M. solution), and in the other cuvette with G-6-P and 6-PG as substrates (0.1 ml. of 0.02 M. solution of each). G-6-P.D. activity is calculated by substracting the value of the former from that of the latter, and in the data to be presented this value has been designated the "derived" G-6~P.D. value. Data on enzyme activity as measured by these two methods are listed in Table II. These results were obtained on four groups of individuals: normal white adult men and women, Negro women who are "intermediate reactors," a group of men "reactors" (one Greek, one Sephardic Jew, and the rest American Negroes), and a group of Negro women "reactors." RESULTS

Clinical groupings and Hematologic Datct.--The clinical features and peripheral blood findings in these patients suggest t h a t t h e y can be divided into three groups. Cases 1, 2, and 3 comprise the first g r o u p ; all are Caucasian boys. In these patients there was a mild to moderate reduction of hemoglobin values, persistent reticulocytosis, and minimal alteration of red cell morphology, with slight macrocytosis and polychromatophilia. The anemia occasionally exacerbated following u p p e r r e s p i r a t o r y infections and during some of these episodes was so severe t h a t transfusions were necessary. A n o t h e r significant clinical event in this group was the occurrence of hemoglobinuria in Case 1 after the

HEMOLYTIC

ANEMIA

329

administration of Phenacetin dmdng an upper respiratory infection. Splenectomy has not been performed in any of these patients. The second clinical group consists of Case 4, an American Negro girl, and Case 5, a Caucasian boy. In these patients anemia became a p p a r e n t soon after birth, and f r e q u e n t transfusions were required to maintain hemoglobin values above 5 Gin. per cent (see Fig. 3). The m a j o r i t y of the erythrocytes in smears of the peripheral Mood were macrocytic and slightly hypochromic, some were t a r g e t shaped, and a few were finely stippled. The retieulocyte counts were moderately elevated and nucleated red cells were always present in tt~e peripheral blood. Following splenectomy ill these two patients there was a m a r k e d rise in the reticulocyte count and, although anemia persisted, frequent transfusions were no longer necessary. Case 6, a 15-year-old Caucasian girl, illustrates the t h i r d v a r i e t y of congenital hemolytic anemia surveyed in this study. This patient experienced severe hemolysis between 21~ and 12 years of age and transfusions were required at 6 to 8 week intervals. H e r course was complicated by frequent convulsive seizures which were only partially controlled b y antiepileptic therapy. Also, fasting blood sugars in the range of 27 to 54 rag. per cent were observed. The red cells showed m a r k e d anisocytosis, poikilocytosis, stippling, and polychromatophilia, and in addition contained inclusion bodies which were demonstrable by the Leishman-Giemsa stain and in wet films of the peripheral blood. An abnormality of pigment metabolism was suggested because of the presence in

330

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the urine of a substance belonging to t h e . bilifuscin and mesobilifuscin groups. S p l e n e c t o m y at 4 y e a r s of age h a d only a t r a n s i e n t beneficial effect on the hemolytic process.

GIutathione Studies and Assays for Glucose-6-Phosphate Dehydrogenase Activity in the Erythrocytes.--The results of these studies are listed in Table I I I . Cases 1, 2, a n d 3 manifested a m o d e r a t e to m a r k e d deficiency of reduced G S H in their erythrocytes, an a b n o r m a l decrease in G S H a f t e r incubation of whole blood with A P H , and a complete deficiency of G-6-P.D. of the erythrocytcs. I n Cases 1 and 3 no reduction of T P N occurred in a 30 minute period even t h o u g h the amount of hemolysate usually employed was doubled. The addition of n o r m a l h e m o l y s a t e to the assay syst e m at this time was followed b y a n o r m a l r a t e of reduction of TPN. G S H values of whole blood and results of the glutathione stability test were n o r m a l in Cases 4 a n d 5. G-6P.D. a c t i v i t y d e t e r m i n e d b y using G-6-P as s u b s t r a t e was elevated. " D e r i v e d " G-6-P.D. values, however, were normal. L o w whole blood G S H values were obtained in Case 6 a n d there was an a b n o r m a l decrease in G S H d u r i n g the G S H stability test. W h e n glucose was a d d e d to blood p r i o r to the GSI~I stability test, the p e r cent of decrease of G S H was less but still g r e a t e r t h a n t h a t observed in normals. G-6-P.D. a c t i v i t y as m e a s u r e d b y b o t h assay techniques was m a r k e d l y elevated.

Family Studies.--Peripheral blood findings and the results of glutathione and enzyme studies on the families of the p a t i e n t s are p r e s e n t e d in Tables I and I I I .

OF P E D I A T R I C S

Cases 1 and 2: The parents of these patients as well as the brother and the m a t e r n a l g r a n d m o t h e r had no abnormalities of their peripheral blood. The maternal g r a n d f a t h e r has had" intermittent jaundice since childhood, and a eholecystectomy was p e r f o r m e d at 40 years of age because of gallstones. When studied in this laboratory, his hematocrit was 35 p e r cent, the reticuloeyte count was 14.3 per cent, and the morphology of the red cells was similar to t h a t observed in Cases 1 and 2. Two brothers of the m a t e r n a l grandfather also had a history of a chronic hemolytic anemia, but their blood was not available for study. Whole blood G S H values, the glutathione stability determinations, and assays for G-6-P.D. a c t i v i t y were norreal in the father, the brother, and the m a t e r n a l grandmother. Results of these studies on blood f r o m the maternal grandf&ther were comparable to those observed in the patients. The mother's glutathione stability test was in the intermediate range, and her G-6-P.D. value as determined with G-6-P as substrate was low normal (Table I I I ) .

Case 3: P e r i p h e r a l blood findings in the p a r e n t s and the b r o t h e r of this p a t i e n t were normal. Glutathione studies on the m o t h e r ' s blood revealed a n o r m a l level of G S t t in her erythrocytes. A f t e r i n c u b a t i n g blood with A P E , however, the resulting decrease in G S I t was similar to t h a t observed in N e g r o w o m e n who are " i n t e r m e diate r e a c t o r s . " The " d e r i v e d " G-6-P.D. value w a s definitely decreased, a l t h o u g h G-6-P.D. a c t i v i t y m e a s u r e d with G-6-P as s u b s t r a t e was low n o r m a l (Table I I I ) .

TABLE I I I . GLUTATHIONE VALUES OF THE ]~HOLE BLOOD~ I~ESULTS OF TtIE GLUTATHIONE STABILITY TEST~ AND LEVELS OF GLUCOSE-6-PHoSP]:IATE ]~)EHYDROGENASE ACTIVITY OF EI~YTIIROCYTES IN PATIENTS WITI-I CONGENITAL NONSPHE]~0CYT]C ]~[EMOLYTIC' A N E M I A

SUBJECT Family 1% Father 2/12/58 4/25/59 Mother 2/12/58 4/25/59 Case

1 2/12/58 4/25/59 Brother 2/12/58

2 2/12/58 Maternal grandfather 4/3/58 3/31/59 Maternal grandmother 4/3/58 3/31/59 Family K Father 11/25/58 4/3/59 Mother 11/25/58 4/3/59 Brother 11/25/58

ENZYME ACTIVITY/ (U,/100 IAs RED BLOOD CELLS) ~EDUCED GLUTATHIONE GLUCOSE-6-PHOS(~o./lOO ~ m ~ED BLOOD CEL~S) PHATE DEIIY~ DERIVED ~ ~ " DROGENASE GLUCOSE-6GLUCOSE (GLUCOSE-6P~-IOSP}I ATE ADDED BEFORE BEFOt%E AFTE~ PHOSPHATE AS DE}IYDt%OGENINCUBATION INCUBATION INCUBATION SUI~STRATE) ASE %VIT~ A P ~ WITH AP~ WITH APH

60 57

45

64

59 41

39

8

62

53

45

14

198 222

146

185 172

]15

0 0

0

226

Case

98

0 0~

1

81 256

162

70 66

49

51 59

258 224

162 132

65 62

3]

36 44

180 166

77 108

56

42

44

272

158

23 28

1

0 7

Case 4 7/15/58 7/16/58 7/23/58 7/25/58 8/1/58

94 64 65 77 67

71 50 53 66 48

69

Case 5 7/24/58

64

53

41 45

28 28

68 74

45 51

Case

3 11/25/58 4/3/59

Family S Father 3/17/59 3/27/59 Mother 3/17/59 3/27/59

0 0~

0 0

300

152

356

188

27 3]

337 3]4

240 215

49 55

210 214

151 163

Case 6 3/17/59 49 ?O 35 411 278 3/27/59 50 27 34 394 239 *Optical d e n s i t y c h a n g e s were followed for a period of 30 m i n u t e s in these hemolysates.

332

THE J O U R N A L OF PEDIATRICS

Cases 4 and 5: Examination of the peripheral blood as well as nonincubated and incubated osmotic fragility studies on the parents of these patients were normal. No abnormalities of glutathione metabolism or enzyme activity were observed in erythrocytes from the mother of Case 4. Case 6: Values for the hematoerit, hemoglobin, and reticuloeyte count in the parents were normal. Wet films of the peripheral blood were examined for inclusion bodies, but none were seen. Glutathione and enzyme studies on the mother's blood were normal. The father had an abnormally low concentration of GStt before and after incubation of,whole blood with APH. Activity of G-6-P.D. in his erythrocytes was markedly increased, although the magnitude of elevation was not as great as that observed in the erythroeytes of his daughter. DISCUSSION

Most of the congenital hemolytic diseases can be classified by alerations in the morphology of the erythrocytes, the electrophoretic pattern of the hemoglobin, and the osmotic fragility of the red cells. There still remains, however, a small number of congenital hemolytic disorders which elude a clear-cut definition by these methods, and to this group has been applied the term "congenital nonspheroeytic hemolytic anemia." Since the diagnosis is made only after certain well-recognized hemolytic disorders have been eliminated, one would expect to find a heterogeneous collection of anemias in the literature under this title. On the basis of differences in red cell morphology, severity of the hemolytic process, and response to splenectomy,

some investigators have indeed suggested that not all patients with this diagnosis are suffering from the same disease2, s Attempts to elucidate the abnormality of red cell metabolism in these patients have also led to the recognition of several different forms. Selwyn and DaMe' described two types after observing changes in cation concentrations, rate of autohemolysis, and osmotic fragility of erythrocytes incubated in vitro. In Type :l, autohemolysis, osmotic, and cation changes on incubation were normal, but the degree of autohemolysis after the addition of glucose was decreased to a lesser extent than that observed in normal blood. In Type 2, the rate of autohemolysis, increase in osmotic fragility, and loss of potassium were markedly increased, and the addition of glucose had no effect on these abnormalities. Furthermore, erythroeytes from these patients utilized glucose at a rate which was 25 to 30 per cent of that observed in normals. Studies o n phosphate metabolism of red cells from 4 patients with congenital nonspherocytic hemolytic anemia were performed by Prankerd. is Two groups were discernible: in one (3 patients) the concentration of adeno~ sine triphosphate, the exchange of s2P~orthophosphate, and the utilization of glucose were decreased; in the other (1 patient) these metabolic parameters were normal. More recently, another type of metabolic abnormality has been described in erythrocytes from patients with chronic nonspheroeytic hemolytic anemia. Newton and Bass 1~ found an instability of glutathione and a deficiency of glucosc-6-phosphate dehydrogenase (G-6-P.D.) in red cells of

ZINKHAM

AND L E N H A R D :

a 4-year-old Italian boy who had a chronic nonspherocytic hemolytic anemia. A n o t h e r publication b y Newton and F r a j o l a 2~r e p o r t e d similar findings on 2 other children, both Caucasian boys, who also were affected with this disease. A familial b a c k g r o u n d for these abnormalities was not d e m o n strable. In the present study a heterogeneous group of 6 patients with congenital nonspherocytic hemolytic anemia could be divided into three categories t h r o u g h glutathione and enzyme studies on the erythrocytes. The first group, comprising Cases 1, 2 and 3, manifested a slight to moderate decrease of whole blood GSH, an instability of GSH a f t e r incubation of blood with API-I, and a deficiency of e r y t h r o c y t e G-6-P.D. In the second group, Cases 4 and 5, these studies were normal ; while in the third group, Case 6, the GSH values before and a f t e r exposure of blood to A P H were m o d e r a t e l y decreased, whereas the activity of G-6-P.D. was greatly increased. Severity of hemolysis and morphology of the red cells also differed in these three groups. In Group 1 the anemia and reticulocytosis were mild, although exacerbatiorr of hemolysis occasionally occurred during infections. The red cells of these patients were normocytic and normochromic. The anemia and degree of reticulocytosis were more m a r k e d in Group 2; the e r y t h r o c y t e s were macrocytic, slightly hypochromic, and peripheral erythroblastemia was usually present, even prior to splenectomy. The one case in Group 3 was unique, for the clinical and hematologic data were quite unlike those

HEMOLYTIC ANEMIA

333

found in other varieties of congenital hemolytic anemia. Since all of these patients have a congenital disorder, one would anticipate that the anomalies of their erythroeytes are genetically determined. Routine hematologic procedures revealed no abnormalities in the peripheral blood from the parents of the patients in Group 1. Glutathione and enzyme values in the m o t h e r of Cases I and 2 and the mother of Case 3, however, were abnormal and were comparable to those observed in Negro women who are " i n t e r m e d i a t e rea c t o r s . " F u r t h e r m o r e , the maternal g r a n d f a t h e r of Cases :[ and 2 had a chronic nonspherocytic hemolytic anemia, and his glutathione and enzyme studies were abnormal (see Fig. 2). These findings suggest that the gene or genes responsible for this v a r i e t y of nonspher0cytic anemia are sex]inked and also indicate that the biochemical alterations of the red cell are a more sensitive index of gene action. In factl the presence of the abnormal gene in the heterozygote woman would be overlooked if routine hematologic procedures were employed as a measure of its phenotypic expression and the gene would be considered impenetrant. Another example of the greater sensitivity of the biochemical m e t h o d in demonstrating the genetic alteration is illustrated in the family of Case 6. E v e n though the morphoL ogy of the red cells and the hematocrit and reticulocyte values were norreal in the parents, the f a t h e r ' s glutathione and enzyme levels were similar t o those observed in the patient. The pathogenetic significance of these metabolic abnormalities has not

334

THE

JOURNAL

been established. The findings in the first group of patients were c o m p a r a ble in some ways to those observed in people with drug-sensitive erythrocytes. The m o r p h o l o g y and fragility of the red cells were unaltered and the genetic transmission of the defect appeared to be the same. Unlike primaquine-sensitive individuals, the hemolytic process in these patients was continuous and not related to the administration of drugs, although in Case 1 hemolysis was accentuated during the administration of Phenacetin. Also, the patients were members of racial groups in which the phenomenon of primaquine-sensitivity has n e v e r been described, and their glutathi, one and enzyme deficiencies were occasionally more pronounced. The level of whole blood GSII in Case 3 and G-6-P.D. activity in Cases 1, 2, and 3 were less t h a n that observed in most primaquine-sensitive individuals. W h e n these enzyme values are comp a r e d to those of a person u n d e r g o i n g a primaquine-indueed hemolysis, the difference becomes more marked. During the course of hemolysis in sensitive individuals, G-6-P.]). activity rises coneomittantly with the reticulocyte count. ~ In patients with this v a r i e t y of congenital nonspherocytie hemolytic anemia, enzyme activity is completely absent in the face of persistent retieuloeytosis. It is suggested, therefore, that the m u t a n t gene responsible for the enzyme deficiency in these patients differs from the one occurring in " p r i m a q u i n e - s e n s l t i v e " people. E v e n though G-6-P.D. activity is not demonstrable, the possibility still exists that this abnormality is not the sole etiologic lesion and that additional genetic or environmental fac-

OF P E D I A T R I C S

tors are necessary for the evolution of the disease. F o r example, the rate of hemolysis in these patients is increased during infections and following the administration of certain drugsY ~ Avoidance of these as well as other unrecognized nongenetic factors m a y lessen the degree of hemolysis. Glutathione and enzyme studies on the patients in Group 2 were normal. Other methods are obviously necessary to demonstrate the inborn error of metaboHsm in the red cells of these patients. The changes observed in Case 6 and her f a t h e r are quite unusual and, unlike the p a t t e r n observed in primaquine-sensitive persons, low GSH values are associated with a m a r k e d increase in G-6-P.D. activity. The results of the GSH stability test in this case indicate the importance of adding glucose du~ing the performance of this procedure. In the absence of glucose, GSH values were 20 and 27 rag. per 100 ml. red blood cells after incubating blood with A P H ; with added glucose, these levels were 35 and 34 rag. per 100 ml. red Mood cells. These findings are p r o b a b l y related to the hypoglycemia which the patient has demonstrated in the past and suggest that the red cell changes may' be a manifestation of a generalized abnormality of carbohydrate metabolism. SUMMARY

The clinical course, hematologic data, and red cell glutathione and glueose-6-phosphate dehydrog e n a s e

studies are r e p o r t e d for 6 patients who had a congenital nonspheroeytie hemolytic anemia. On the basis of these findings the patients could be divided into three groups.

ZINKHAM AND LENHARD:

HEMOLYTIC ANEMIA

335

Group 1 consists of three Caucasian paths for exploration are becoming boys belonging to two families. The apparent. When these pathways are anemia and reticulocytosis were mild, finally mapped, therapeutic application red cell morphology was normal, red of this knowledge may become possible. cell glutathione was unstable in the REFERENCES presence of acetylphenylhydrazine, 1. Beutler, E.: The Hemolytic Effect of and glucose-6-phosphate dehydrogenase F r i m a q u i n e and Related Compounds: A activity was absent. Glutathione and Review, Blood 14: 103, 1959. enzyme studies on the family members 2. Daei% J. V., ~/[ollison, P. L., Richardson, N., Selwyn, ft. G., a n d Shapiro, L.: indicated that these abnormalities Atypical Congenital tiemolytiz Anemia, were genetically determined and that Quart. J. Med. 22: 79, 1953. 3. Thompson, W . P . : Itemolytic Jaundice, their transmission was sex-linked. Bull. New York Aead. 1Ked. 15: 177, A Caucasian boy and a Negro girl 1939. 4. Crosby, W. H.: H e r e d i t a r y Nonspherocomprise Group 2. The anemia and cytie Hemolytic Anemia, Blood 5: 233, reticulocytosis were quite marked, 1950. 5. Kaplan, E., and Zeulzer, W. W.: Faand the erythrocytes were macrocytic, milial Nonspherocytic Hemolytic Aneslightly hypochromic, and occasionmia, Blood 5" 811, 1950. ally target shaped and finely stippled. 6. Haden, R . L . : A New Type of Heredit a r y Hemolytic J a u n d i c e W i t h o u t Nucleated red cells were usually presSpheroeytosis, Am. J. M. Sc. 214: 255, ent in the peripheral blood. Glutathi1947. 7. Feinberg, A. W., and Watson, J.: Nonone and enzyme studies were normal. spherocytic Chronic Hemolytic Anemia Group 3 consists of a 15-year-old W i t h Basophilic Stippling. Report of a Case in a' Negro, Blood 6: 357, 1951. Caucasian girl who was first described 8. Motulsky, A. G., Crosby, W. H., and by Lange and Akcroyd. n She had a Rappaport, H.: H e r e d i t a r y Non-spherocytic HemoIytle Disease. A S t u d y of a severe hemolytic anemia between 21/2 Singular Familial Hemo]ytlc Syndrome, and 10 years of age, and inclusion Blood 9: 749, 1954. 9. Selwyn, J. G., and Dacie, J . V . : Autobodies were observed in the red cells. hemolysis a n d Other Changes Resulting Frequent epileptic seizures and blackFrom the I n c u b a t i o n I n Vitro of Red Cells From P a t i e n t s W i t h Congenital colored urine were also noted. Whole Hemolytic Anemia, Blood 9: 414, 195t. blood glutathione values and the glu- 10. Newton, W. A., Jr., a n d Bass, J. C.: tathione stability test were abnormal. GIutathione-Sensitive C h r o n i c Nonspheroeytie Hemolytic Anemla~ A. :M. Erythrocyte glueose-6-phosphate deA. J. Dis. Child. 96: 501, 1958. hydrogenase activity was increased. 11. Lange, 1~. D., and Akeroyd, J. It.: Congenital Hemolytic A n e m i a W i t h AbnorThe father's peripheral blood count mal P i g m e n t 3/[etabolism and Red Cell was normal, but his glutathione and Inclusion Bodies: A New Clinical Syndrome, Blood 13: 950, 1958. enzyme values were similar to those 12. Ham, T . H . : A Syllabus of L a b o r a t o r y observed in the patient. E x a m i n a t i o n s in Clinical Diagnosis, These studies emphasize the importance of biochemical techniques in shedding light on the mechanism of hemolysis and the pattern of inheritance of congenital hemolytic disorders. Even though our knowledge of the etiologic role of many of these metabolic aberrations is incomplete, newer

Cambridg% 1957, t I a r v a r d U n i v e r s i t y Press. 13. Beutler, E., Dern, R. J., F l a n a g a n , C. L., and Alving, A . S . " The Hemolytic Effect of Primaquine. VII. Biochemical Studies of Drug-Sensitive Erythrocytes, J. Lab. & Clin. lV~ed. 45: 286, 1955. 14. Beutler, E.: The Glutathione I n s t a b i l i t y of Drug-Sensitlve Red Cells: A New Method for the I n Vitro Detection of Drug Sensitivity, J. Lab. & Clin. Med. 49: 84, 1957.

336

THE JOURNAL OF PEDIATRICS

!5. Childs, B., Zinkham, W. tI., Browne, E. A., Kimbro, E. L., and Torbert, J. V.: A Genetic Study of a Defect in Glutathlone /Vletabo]ism of t h e E r y t h r o c y t e , Bull. J o h n s Hopkins Hosp. 102: 21, 1958. 16. Glaser, L., and Brown, D . H . : Purification and P r o p e r t i e s of D-glucose-6-phosp h a t e Dehydrogenase, J. Biol. Chem. 216: 67, 1955. 17. Zinkham~ W. H.: An I n Vitro Abhorm a l i t y of Glutathione Metabolism in E r y t h r o c y t e s From Normal Newborns: 1Viechanism and Clinical Significance, Pediatrics 23: 18, 1959. 18. Glock, G. E., and :~r P.: F u r t h e r Studies on the Properties and Assay of Glucose-6-phosphate Dehydrogenase and

6-phosphogluconate Dehydrogenase of R a t Liver, Biochem. J. 55: 400, 1953. 19. P r a n k e r d , T. A. J.: I n b o r n Errors of lVIetabolism in Red Cells of Congenital Hemolytic Anemia, Am. J. IVied. 22: 724, 1957. 20. Newton, W. A., Jr., and Frajola, W. J.: Drug-Sensltive Chronic Hemolytic Anemia: F a m i l y Studies, Clln. Res. 6: 392~ 1958. 21. F l a n a g a n , C. L., Schrier, S. L., Carson, P. E., and Alving, A. S.: The ttemolytlc Effect of Primaquine. V I I I . The Effect of Drug Administration on Parameters of P r i m a q u i n e Sensitivity~ J. Lab. & Clin. ]~ed. 51: 600~ 1958.