ACTIVITY OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE IN ERYTHROCYTES OF PATIENTS WITH VARIOUS ABNORMAL HEMOGLOBINS

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ACTIVITYOF GLUCOSE-6-PHOSPHATE

DEHYDROGENASEIN

ERYTHROCYTES

OF PATIENTSWITH VARIOUS

ABNORMAL HEMOGLOBINS

Jean Naylor, M.D., Ira Rosenthal, M.D., Aaron Grossman, M.D., Irving Schulman, M.D., and David Yi-Yung Hsia, M.D.

Departments of Pediatrics, Northwestern University Medical School, University of Illinois Medical School, and Chicago Medical School; the Children's Memorial Hospital, the University of Illinois

Research and Education Hospital, and the Children's Division of the Cook County Hospital, Chicago

W HEN primaquine and certain other

ordinarily harmless drugs are admin istered to some individuals, generally of

Negro or Near Eastern extraction, an acute

hemolytic anemia results.13 These patients have an erythrocyte abnormality charac terized by low levels of reduced glutathi one,4 marked reduction in these levels when whole blood is incubated with acetyiphenyl hydrazine,@ and low activity of

glucose-6-phosphate dehydrogenase.Â° The defect is

hereditary in nature and transmitted by a

sex-linked gene of intermediate domi nance.2'7'8

The incidence of pnimaquine sensitivity

tends to be high in areas where there is also

high incidence of sickle cell disease. This has been demonstrated by field surveys of

isolated population groups in Africa and Sardinia.9

The purpose of the present paper is to

report on the occurrence of the two condi

tions in the same individual, to observe

whether this alters the clinical pattern of

sickle cell anemia, and to compare the in cidence of decreased activity of

glucose-6-phosphate dehydrogenase in erythrocytes of

Negro patients with sickle cell anemia and those with normal hemoglobin.

CLINICAL MATERIAL AND METHODS

The present survey was undertaken on a total

of 99 patients with sickle cell anemia (5-5), 11 patients with sickle cell-hemoglobin C disease (S-C), 6 patients with sickle cell-thalassemia disease (S-Th), and 56 individuals with sickle cell trait (A-S). A group of 100 hematologically normal Negroes of similar age served as con trols.

A total of 10 ml of hepaninized blood was ob

tained from each individual. The diagnosis of the hemoglobinopathies was made by paper electrophoresis. The concentration of gluta thione (GSH) and the stability of CSH in the presence of acetylphenylhvdrazine (APH) was determined by the method of Beutler.5 Al though most of the determinations were meas ured within 2 hours after collection of the blood, some specimens were kept overnight with 3 mg/ml of added glucose. This was done in about a fourth of the patients with S-S, half of the individuals with A-S, and a fourth of the controls. There was no significant difference in the range of the values obtained from those analyzed within two hours and those kept overnight with glucose. The results were ex pressed as mg/100 ml of packed enythnocvtes

(RBC).

Activity of glucose-6-phosphate dehydroge

nase (G6PD) was determined as described by Zinkham.1Â° Readings were made at 340 m@ at

5 to 15 minutes and expressed as enzyme

units per 100 ml of packed erythnocytes. If a

reading could not be obtained at 340 m@i.,the

specimen was diluted and read. Enzyme activ ity was also expressed as L@O.D. pen minute pen gram of hemoglobin for many specimens.

With technicalassistanceof Chui-Yee Wong, M.S.

Dr. Naylor was a Research Fellow of the Schweppes Foundation and the Spastic Paralysis Research Foundation.

This work was supported by grantsfrom the Otho S.A. Sprague Memorial Institute,and the Chicago Community Trust.

ADDRESS:(D.Y.H.) Children's Memorial Hospital, 707 Fullerton Avenue, Chicago 14, Illinois.

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AA CONTROLS FEMALES PRE â€”¿INCUBATION MEAN 604 SD Â±54

POST INCUBATION MEAN 50 SD tb

(mg/bOOst) GSH (mg/IOOmI)

SS HEMOGLOBIN PREâ€”INCUBATION MEAN 729 Â±212 POST INCUBATION MEAN 564 Â±168

GSH

0 20

RESULTS Reduced Glutathione

Among control nonreactors (defined in

next paragraph), the initial level of GSH

prior to incubation with APH was 60.4 Â±

15.4 mg/100 ml RBC; 63.1 Â± 12.8 mg/100 ml RBC among those with A-S hemoglobin,

and 72.9 Â± 21.2 mg/100 ml RBC among

those with S-S hemoglobin. The differences are not significant, although the range among those with sickle cell anemia appears to be greater.

Among control reactors, the initial level

of GSH was 45.5 Â± 14.1 mg/100 ml RBC;

47.1 Â± 7.5 mg/100 ml RBC among those

with A-S hemoglobin, and 46.4 Â± 13.1

mg/100 ml RBC among those with S-S hem oglobin.

The levels were unrelated to the sex of the individuals tested.

A1rEI@ INCUBATION WITH APH: Follow

ing the incubation with APH, the values for GSH male controls followed the well-known bimodal distribution seen among Negroes (Fig. 1). Of the 56 males 8 (14%) showed a

50% or greater decrease in GSH to below

30 mg/100 ml RBC after incubation with

APH; these were classified as reactors. Of

the 44 females 4 (9@) showed a 50% or

greater decrease in GSH to below 30 gm/

100 ml RBC after incubation with APH;

these were classified as reactors. The mean GSH value after incubation was 50.0 Â±10.1

mg/100 ml RBC for nonreactors and 12.0

Â± 6.4 mg/100 ml RBC for reactors.

Among the individuals with S-S, 8 out of 54 males (15%) and 4 out of 45 females (8.9%) were found to be reactors (Fig. 1). The en tire group showed a wider distribution of values, although the mean values of 56.4 Â± 16.8 mg/100 ml RBC for the nonreactors and 13.9 Â±8.3 mg/100 ml RBC for the re

MALES

8â€”

6â€” 4â€”

2â€” Ii

20 40 60 80

GSH (m,/IOOmI) boo

PRE â€”¿

POST

0 20 40 60 so too

GSH (mg/IOOmI)

S

4.

II

I t @F

so so 100

GSH (mg/bOOml)

8

eG SO bOO

OSH Img/lOOmI)

FIG. 1. Results of the glutathione stability test in the erythrocytes of patients with normal

hemoglobin, S-S hemoglobin and A-S hemoglobin. The results are expressed as mg/100

ml packed erythrocytes.

0-8â€”

6- 4-

2-0

8â€”

6â€”

(3)

I . I I T

bOO 200 300 400 500 600 700

6-6â€”PD (units pir bOOml/RBC) 0

0 bOO 200 300 400 500 600 700

6â€”6â€”PD (unIt. pr IOOml/RBC)

actors are not significantly different from the control subjects. Of the 56 individuals with A-S 17 (30%) were reactors. This high incidence among those with sickle cell trait is accounted for by the fact that many of these were found among the families with

members who had reduced G6PD in their

erythrocytes.

In addition, four patients with S-C and

one patient with S-Th were found to be

reactors.

Glucose-6- Phosphate Dehydrogenase

In general, individuals whose erythro cytes had GSH levels below 30 mg/100 ml

RBC after incubation with APH had G6PD

levels below 100 units/100 ml RBC. The

only exceptions were in four females (in cluding one control, two A-S and one S-S)

whose erythrocytes had high enzyme values

and post-incubation GS'H values between

20 and 30 mg/100 ml RBC. Also, in one

female S-trait the enzyme value was below 100 units/100 ml RBC, but the post-incuba

tion GSH value was 38 mg/100 ml RBC. Among the nonreactors, the G6PD levels

in units/100 ml RBC were 202 Â± 47 for con

trols, 204 Â±48 for A-S, and 3q51Â±109 for

S-S. The difference between the latter

and the other two groups is significant (P<0.001). The range of G6PD activity of

the reactors and nonreactors is shown in Figure 2. Erythrocytes from patients with S-C had mean values of 275 Â±81 units and from those with S-Th, 345 Â±95 units/100 ml RBC.

Although the number of reactors was

small, the same trend could be noted. The

G6PD levels were 21 Â± 19 units/100 ml

RBC for controls, 27 Â± 25 for A-S, and

36 Â±28 for S-S.

Because of the possibility that the shape of the sickle cell played a role in these dif ferences, the enzyme activity was expressed

as @O.D.per minute per gram of hemo

globin (Fig. 3). It can be seen that the dif ference still appears.

In the group with the sickle cell anemia

MALES _AA _CONTROLS _FEMALES

MEAN 2026 SD 47 Â±

JL

I I U I 1 1

.0 200 300 400 500 600 700 800 6-6 - PD (units p.r 100 ml/ RBC)

..A1.L...

_I _I _-@ â€”¿

0 bOO 2@)0 300 4@0 500 600 700 6â€”6â€”PD(units p.r bOOmb/RBC)

AS HEMOGLOBIN

MEAN 2042 SD 48 Â±

0 100 200 300 400 500 600 700 800

6â€”6â€”PD (units psr IOOmI/RBC)

Fic. 2. The activity of glucose-6-phosphate dehydrogenase expressed as units/100 ml packed erythrocytes in the same population as Figure 1.

SS HEMOGLOBIN

MEAN 35b SD 097 Â±

7 6 5 4 3 2

I I U â€”¿I U -@

0 bOO 200 300 400 500 600 700 800

(4)

700

600

S

vs

6) -@ 500

U S â€¢¿ B

2 5 S

-C

-@.. 400 5 1

6) 5 5 S

0 8 S S

@ HEMOGLOBIN

-@ â€¢¿I@ S

0 g

@ ₂ ₃₀₀ _@;s S

@ 0 AS HEMOGLOBIN

%@@% @D S@+

@ S â€¢¿ + AA HEMOGLOBIN

vs 0,4S S

:@ :Ã¸.it @:p4â€¢â€¢+

:D 200 6

@ ,

4@v. . S

@ *@.4 0

too 5+ â€¢¿

+

0 @

@ I I I I I I I I

4 8 2 16 20 24 28 32 36 40 44 48 52

L@O.D./min/gm hemoglobin

Fic. 3. The activity of the glucose-6-phosphate dehydrogenase expressed in units! 100 ml of packed erythrocytes compared with activity expressed as @O.D./min/gm of hemoglobin.

700-600

vs

6) 500

U 2

400

6)

I

-@

300

vs

C 200

D

100

I I I I I I I I I I

I 2 3 4 5 6 7 8 9 0 II 12 13 14 IS 16 Ii 18

Age (years)

(5)

Units/lOO ml erythrocytes

KEY

Ss .

Cs

S thol+

Fic. 5. Relationships of reticulocyte count and activity of glucose-6-phosphate dehydro genase (expressed as units! 100 ml packed erythrocytes).

. .

U)

6)

U

0

6) 0 0

I,,

6)

U

0

U

6)

â€¢¿

0 0

.

Sâ€¢ S

S

â€¢¿S

.â€˜ S

â€¢¿

0-â€˜¿S

0 bOO

S

200

â€¢¿

300 400 500

â€¢¿

S â€¢¿

â€¢¿.(@

600

â€¢¿

700

â€¢¿

0

S â€¢¿

â€¢¿

0

in which high values were encountered, no correlation was found between G6PD activ ity in the erythrocytes and the degree of anemia or age of the patient (Fig. 4). Simi larly, there was no correlation between the enzyme activity and the reticulocyte counts (Fig. 5).

Family Studies

Family studies demonstrated conclusively that the two defects segregated independ ently in 14 families presenting both defects

(Figs. 6,7).

In the course of these studies, four

mothers of affected males were encountered whose erythrocytes had normal post-incu bation GSH levels and normal G6PD levels. In one of these families, the father was a reactor. However, since other fathers in af fected families have also been shown to be reactors, this probably was a chance oc

currence. Rather, one is led to believe that

the penetrance for the trait is variable in the female, as has been reported.7'8'1'

Clinical Course

The clinical course of those individuals

with hemoglobinopathies and the enzymatic

defect was compared with the course of pa tients having hemoglobinopathies without

the enzymatic defect. No significant differ ences were found in the number of crises,

growth, degree of anemia or occurrence of unusual manifestations. One reactor with sickle cell anemia had evidence of liver disease and one reactor with sickle cell hemoglobin C disease had evidence of renal disease. One 9-year-old boy with both low

enzyme activity in the erythrocytes and

sickle cell anemia had an unusually mild

course; he had no complaints until he was

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PalieniType of Heino

globinActivity

of Glucose 6-p/zosphaie Dc hydrogeno.se and

Glutathione StabilityErythroc$e

Halfife in Days (Cr6' Method)L.J.

J.A. C.M. H.C. L.S. L.P.S-S

S-S

S-S S-S

S-C

S-CNormal Normal Low' Low' Normal

Low'74

7 7

6

14 1@ LjJAA cjas

AS

AS @AS @as @5J AS

role

6-6â€”PS s sos u.@.. t APO

0â€”100 + )S0% P00â€”ISO + 0 50 S

.00,150 + (00%

Fic. 6. Genetic segregation of S-hemoglobin and deficiency of glucose

6-phosphate dehydrogenase in the erythrocytes in families with sickle

cell anemia.

â€¢¿

.

ci

@

Do

studies with Cr'1 were performed.12 The survival time of the erythrocytes of six pa tients is shown in Table I. The coexistence of the two abnormalities in the erythro cytes did not affect the shortened survival time usually found in these patients. The erythrocytes of two nonreactors with sickle cell anemia had half-lives of 7% and 7 days, and cells from two reactors with sickle cell anemia had half-lives of 7 and 6 days. Sim ilarly, survival of erythrocytes from two pa tients with S-C disease (one a reactor and one a nonreactor) were not significantly dif ferent.

DISCUSSION

It has been demonstrated by this study that deficiency of G6PD activity in the erythrocytes of some patients with S-S dis ease is not responsible for the known vari ability of the course and symptoms of the

disease. It is probable that no increase in

Erythrocyte Survival

Even though there were no differences in the clinical course, the possibility that the combined defect in the erythrocytes could lead to a decreased life span of the erythro cytes was considered. Erythrocyte survival

TABLE I

SUIWIvAL OF ERYTHROCYTESIN RELATION TO OTHER FACToRS

â€˜¿Seetext for definition.

L1JAS Â¶AS Â¶as Â¶AS

Fom+yC

@ F@IyA

@

LjJAS @AS

FonolyM

Â¶

OS SO AS AA LA AA

(7)

C+S

@AC

A+ S

B Fomlby

severity of S-C disease or S-Th disease will be found as a result of a similar combination of defects.

In patients with primaquine sensitivity, it has been shown that for a period after an acute hemolytic episode they are relatively refractory to further hemolysis when the

drug is administered. This has been at

tributed to the young population of erythro cytes. In the erythrocytes of reactors with sickle cell anemia there exists an abnormal GSH stability and low G6PD as well as there

being a young population of erythrocytes.

This may exert a protective effect. Studies to

determine whether the erythrocytes in these

patients are sensitive to primaquine are in progress.

The association of high enzyme levels and normal GSH stability in erythrocytes of pa tients with sickle cell anemia might be ex pected in the presence of a younger popu lation of cells.'3 The lack of correlation of reticulocyte count with enzyme value does not eliminate this possibility, as the reticulo

P Family $ Family N Family F Family

AS TAS

6-6-PD/bOmb RBC

0â€”100 100 â€”¿150 over ISO NOT TESTED

S OSH dicr.

@ APH

+ )50S

+ )50%

+ (50%

cytes are not a complete indication of cell

maturity.

Finally the family studies indicate that, for both controls and sickle cell disease, the gene expression in the female appears to be quite variable. The heterozygote can some times be detected by either the GSH-stabil ity test or the level of G6PD alone. Despite using both tests in the case of several mothers of male reactors it was not possible to show abnormal reduction of GSH levels after incubation with APH or low values for G6PD upon repeated testing.

SUMMARY

In 99 patients with sickle cell anemia and

100 normal Negro children, the activity of

glucose-6-phosphate dehydrogenase in

erythrocytes was determined.

Hemoglobinopathies may be combined

with reduced activity of glucose-B-phos phate dehydrogenase in the erythrocytes.

The two defects are inherited independ Fic. 7. Genetic segregation of S- and C-hemoglobins and deficiency of

glucose-6-phosphate dehydrogenase in the erythrocytes in affected families.

ently.

MALE FFMALE

U

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292

The incidence of the enzymatic defect in the erythrocytes of those with sickle cell anemia in this series does not differ from that of a similar Negro population having normal hemoglobin.

There appears to be no significant effect of the enzymatic defect in the erythrocytes on the clinical course of patients with sickle cell anemia or survival times of their eryth rocytes.

Patients with sickle cell anemia and with normal stability of the glutathione in the

erythrocytes have significantly higher levels

of GGPD than controls or patients with

sickle cell trait.

Acknowledgment

We wish to thank Dr. Aaron Josephson,

Michael Reese Hospital, for allowing us to

study many of his patients, and Dr. H. Harris

for his suggestions.

REFERENCES

1. Hochwald, R. S., Arnold, J., Clayman, C. B., and Alving, A. S.: Status of pri maquine. IV. Toxicity of primaquine in Negroes. J.A.M.A., 149:1568, 1952. 2. Szeinberg, A., Sheba, C. H., and Adams,

A.: Selective occurrence of glutathione instability in red blood corpuscles of various Jewish tribes. Blood, 13:1043, 1958.

3. Zinkham, W. H., Lenard, R. E., Jr., and Childs, B. A.: Deficiency of glucose-6-phosphate dehydrogenase activity in erythrocytes from patients with favism. Bull. Johns Hopkins Hosp., 102:169,

1958.

4. Beutler, E., Dens, R. J., Flanagan, C. L.,

and Alving, A. S. : The hemolytic effect of primaquine. VII. Biochemical studies of drug-sensitive erythrocytes. J. Lab.

& Clin. Med., 45:286, 1955.

5. Beutler, E. : The glutathione instability of drug-sensitive red cells. A new method

for the in vitro detection of drug sensi

tivitv. J. Lab. & Clin. Med., 49:84, 1957.

6. Carson, P. E., Flanagan, C. L., Ickles, C. E., and Alving, A. S. : Enzymatic defi

ciency in primaquine-sensitive erythro

cvtes. Science, 124:484, 1956.

7. Childs, B., and Zinkham, W. H. : A genetic study of a defect in glutathione me

tabolism of the erythrocvtes. Bull. Johns Hopkins Hosp., 102:21, 1958.

8. Gross, R. T., Hurwitz, R. E., and Marks, P. A.: A hereditary enzymatic defect in

erythrocyte metabolism: glucose-6-phosphate dehvdrogenase. J. Clin. In vest., 37:1176, 1958.

9. Motulskv, A.: Personal communication.

10. Zinkham, W. H.: An in-vitro abnormality of glutathione metabolism in erythro

cytes from normal newborns: mechanism

and significance. PEDIATRICS, 23:18,

1959.

11. Szeinberg, A., and Sheba, C.: Hemolytic

trait in Oriental Jews connected with a hereditary enzymatic abnormality of

ervthrocvtes. Israel M. J., 17:158, 1958.

12. Erlandson, M. E., Schulman, I., and Smith, C. H.: Studies on congenital hemolvtic syndromes. II. Rates of destruction and production of erythrocytes in thalas semia. PEDIATRICS,22:910, 1958. 13. Marks, P.: Red cell glucose-6-phosphate

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1960;26;285

Pediatrics

Jean Naylor, Ira Rosenthal, Aaron Grossman, Irving Schulman and David Yi-Yung Hsia

HEMOGLOBINS

ERYTHROCYTES OF PATIENTS WITH VARIOUS ABNORMAL