The hemolytic process of viral hepatitis inchildren with normal or deficient glucose-6-phosphate dehydrogenase activity

422

September, 1970 T h e Journal o[ P E D I A T R I C S

The hemolytic process of viral hepatitis in chiMren with normal or deficient glucose-6-pboqohate dehydrogenase activity The incidence and degree o[ the hemolytic process associated with viral hepatitis was studied in 125 children; 16 were deficient in erythrocyte G-6-PD and 5 were heterozygotes for ~-thalassemia, Hemolysis was observed in 24 (23 per cent) o[ the nondeficient subjects, 14 (87 per cent) o[ those with G-6-PD deficiency, and in 4 (80 per cent) o[ the heterozygotes. In the nondeficient patients hemolysis was unusual and mild. Moderate and severe hemolysis occurred only in 6 children with G-6-PD deficiency and in 3 with thalassemia trait. Bilirubinemia was generally more pronounced in the patients with the erythrocyte dejects; levels higher than 25 rag. per cent were noted in 4 o[ them. These findings suggest that either G-6-PD deficiency or thalassemia trait may modi[y the clinical course of viral hepatitis by [avoring the induction of hemolysis and hyperbilirubinemia.

C. A. Kattamis, M.D.,* and F. Tjortjatou, M.D. ATHENS, OREECE

G z o c o s E-6-phosphate d e h y d r o g e n a s e (G-6-PD) deficiency has been incriminated in the pathogenesis of drug-induced hemolytic anemia, favism, and severe neonatal jaundice unrelated to rhesus or ABO incompatibility? It has also been suggested that infectious diseases, especially of viral origin, may produce acute hemolysis in individuals deficient in G-6-PD3 The relationship of an infectious agent to the frequency and the severity of the hemolytic process From the Choremis Research Laboratory, Department of Pediatrics, School of Medicine, Athens University. Supported in part by the Royal Research Foundation Grant 743 *Address: Department o] Pediatrics, University ol Athens, St. Sophie's Children's Hospital, Athens, 608, Greece. VoI. 77, No. 3, pp. 422-430

has not been clearly defined. Hemolytic episodes associated with bacterial infections (mainly pneumonia), with viral upper respiratory tract infections, and with both acute and chronic hepatitis have been reported in G-6-PD-deficient Negroes?, 4 In a preliminary report we showed that hemolysis may also occur in children with the Mediterranean variant of G-6-PD deficiency, during the course of viral hepatitis.5, s In an attempt to clarify certain aspects of the hemolytic process noted in G-6-PD-deficient individuals, the study has been extended to evaluate: (1) the incidence, degree, and pathogenesis of anemia found in the course of infective hepatitis in children, and (2) the difference in the he-

Volume 77 Number 3

Hemolytic process o[ viral hepatitis

molytic process between patients with normal G-6-PD activity and those with deficiency of this enzyme. MATERIAL

AND METHOD

OF STUDY A total of 125 children, aged 2 to 14 years, with infective hepatitis were studied. The diagnosis was based on the presence of clinical and laboratory findings, especially increased serum glutamic oxaloacetlc transaminase and serum-glutamic pyruvic transaminase activity, impaired liver function tests, and hyperbilirubinemia of both unconjugated and conjugated types. In all patients, G-6-PD activity, hemoglobin, hematoerit, red cell morphology, and reticulocyte count were determined. In most patients red cell count and osmotic fragility were also studied. These estimations, except that for G-6-PD, were repeated every 4 to 5 days for a period of 10 to 20 days depending on the severity and the course of the disease. According to the levels of G-6-PD activity, patients were divided into two main groups, the nondeficient and the deficient group. Group I (a, b, c) consisted of 109 children with normal G-6-PD activity. Of these, 51 were boys (Subgroup a), 53 were girls (Subgroup b), and 5 were male or female heterozygotes for fi-thalassemia (Subgroup c). Group II (a, b) included 16 patients deficient in erythrocyte G-6-PD. Of these, 11 were boys (Subgroup a) and 5 were girls (Subgroup b). The presence of a hemolytic or aplastic process was determined on the basis of criteria listed in Table I. The criteria, though somewhat arbitrary, were useful as a tool for analysis. Drugs were restricted from all patients, except those with severe and prolonged jaundice, who received corticosteroids for a short period of time. METHODS

Routine" laboratory methods were used for the determination of bilirubin, liver function tests, and transaminasesY Hemo-

423

Table I. Criteria for establishing the presence of hemolytic or aplastic process

Type and severity of syndrome Hemolytic None Mild Moderate Severe

Aplastic Mild Moderate

Drop in hemoglobin (Gm./lO0 ml.)

Reticulocyte count (%) (Highest values)

< 1.0 1.1-2.0 2.1-3.0 > 3.1

<3 3.1-5 5.1-10 > 10

2.0-3.0 > 3.1

0.5-1 <0.5

globin was estimated as cyanmethemoglobin, hematocrit in capillary tubes, and reticulocytes after incubation of erythrocytes in brilliant cresyl blue solution for two hours at 37 ~ C. with counting of more than 500 cells. G-6-PD activity was tested by the brilliant cresyl blue dye test of Motulsky and Campbell-Kraut. The standards of our laboratory for the brilliant cresyl blue test are: decolorization in 30 to 70 minutes = normal enzymatic activity, 70 to 150 minutes = intermediate or partial deficiency; no decolorization in 150 minutes = complete deficiency.8 In patients with prolonged deeolorization time, G-6-PD activity was also determined by the quantitative method of Zinkham and associates,~ slightly modified; for this method our standards for normality are 394 + 61 (280 to 520 units) per 100 ml. packed red cells. 8 Red cell osmotic fragility was tested in 0.36 per cent buffered NaC1 solution. Osmotic fragility was considered decreased when less than 80 per cent of red cells were hemolyzed. According to the percentage of unhemolyzed cells, the osmotic fragility was graded as greatly (3+), moderately (2+), or mildly (1+) decreased. In three patients the osmotic fragility curve was studied by the method of Parpart and associates?~ Patients suspected of having thalassemia trait were further investigated by starch gel electrophoresis; quantitative estimation of hemoglobin A2 was done using cellulose acetate electrophoresis.

424

Kattamis and Tjortjatou

The Journal o[ Pediatrics September 1970

~

13

FEMALES

C] NON-DEFICIENT

11

I~ G-6-PD DEFICIENT

9 IZ L~

7 5 3

% IHIIIIt

Va. t, 0

f l 1 I I I I 1 IXD~ 19

17=

0r

ta

15

X

13

x

11

txl

MALES

j

9

7 5

3 1

-I-1 l-lll

I

|111

I

IJ I

I

35 45 55 65 75 85 95 10~ . . . . . DECOLORIZATION

140)150

IN

MINUTES

Fig. 1. Results of brilliant cresyl blue decolorization test. In brackets are units of G-6-PD activity per 100 ml. packed red cells,

HEMOLYTIC MILD

SYNDROME

I[MOOERATE II SEVERE elQ5 %

5

8

,4

&11% r.

9 1~,%

W z

9o

&&

0

n 0 P,

&o I I

A O0

X

0

~ X

o 1

2

3

4

RET I C U L O C Y T E S

5

:%

%

Fig. 2. Reticulocyte levels ( % ) a n d fall in hemoglobin values (gram per 100 ml.) of 125 patients. A ~- normal male; A = deficient male; Q) ~ normal female; 9 = deficient female; x -~- fl-thalassemia trait.

Volume 77 Number 3

I~ESULTS G-6-PD activity. The results of the brilliant cresyl blue dye test on 125 patients (64 males and 61 females) are illustrated in Fig. 1. In 92 children (73.4 per cent), decotorization of brilliant cresyl blue was completed in 40 to 60 minutes. Eleven boys were completely defcient. Four girls had intermediate decolorization times (75 to 120 minutes); one had no decolorization after 150 minutes. G-6-PD activity in these girls ranged from 17 to 45 per cent of the normal mean, a range seen in heterozygotes with the Mediterranean variant. Homozygotes are usually totally deficient, s Hemoglobin and reticulocytes. Fig. 2 illustrates the highest level of reticulocytes and the total reduction of hemoglobin in grams per 100 ml. (maximum-minimum level) observed during the course of infectious hepatitis in each of i25 patients. In Group Ia (nondeficient boys) no signs of hemolysis were found in 43 (84 per cent). In the remaining patients in this group only minor evidences of hemolysis were noted. In contrast, in Group I I a (G-6-PD-deficient boys), 10 (91 per cent) had a hemolytic process which was mild in 5, moderate in 3, and severe in 2. The difference in the incidence of hemolytic process between Groups Ia and IIa is statistically significant (X2 = 16.73, p < 0.001). Group Ib (nondeficient girls) had minor differences in hemolysis compared to Group Ia. In 37 (70 per cent) no hemolysis was detected; in the remaining 16 (30 per cent) who had some degree of hemolysis, only a single instance of moderate hemolysis was noted. Of 5 patients in Group IIb (heterozygotic females), 4 (80 per cent) experienced hemolysis, which was severe in one. Hemolysis was also found in 4 of 5 patients w i t h fl-thalassemia trait (Group

Ic). Generally, the incidence of hemolysis was significantly higher in G-6-PD-deficient children (Group II) than in nondeficient ones (Group I) (X2 = 21.59, p < 0.001). Spectrum of hemolytic process. Fig. 3 illustrates the mean, standard deviation, and

Hemolytic process o[ viral hepatitis

425

range of the lowest hemoglobin and the highest reticylocyte and total bilirubin levels observed during the course of hepatitis in the five subgroups. Individual values for Subgroups Ic, IIa, and IIb are also recorded. The mean hemoglobin levels were somewhat higher in Groups Ia and Ib than in Groups IIa and IIb, but the difference was not statistically significant (t = 1.47, p 0.2). The variations in the retieulocyte counts were more impressive. The mean value of reticulocytes for Group Ia was 2.2 +- 1.1 per cent (0.8 to 5 per cent) ; for Group Ib, 2.6 + 1.1 per cent (1.2 to 6 per cent); for Group IIa, 6.1 + 4,4 per cent (2.1 to 18 per cent) ; and for Group IIb, 4.4 + 2.5 per cent (1.2 to 10.5 per cent). The difference in reticulocyte levels is significant between Groups Ia and I I a (p < 0.02) and generally between Groups I and II (p < 0.05). The spectrum of the hemolytic process in Group II varied widely, as indicated by the extreme range of reticulocyte counts, 2.1 to 18 per cent, compared to 0.8 to 6 per cent in Group I. In three G-6-PD-deficient patients reticulocyte values were very high ( > 10 per cent) and hemoglobin values declined more than 3 grams per 100 ml. (Figs. 2 and 3). Bilirubin levels. As indicated by the mean as well as by the wide range of individual values, bilirubinemia was more pronounced in patients with G-6-PD deficiency (t ----- 3.4, p < 0.01). Extremely high levels ( > 30 rag. per cent) were observed only in three G-6PD-deficient patients, two boys and one girl, and in one patient heterozygotic for fi-thalassemia, in whom a bilirubin level of 27.5 rag. per cent was found. To determine the probable effect of hemolysis in increasing bilirubin levels we: (1) compared the bilirubin levels of the patients with and without hemolysis in the two major groups (Ia and Ib); no significant differences were found (Fig. 3); (2) estimated the ratio of unconjugated to total bilirubin in all patients (Table II); no difference was noted in this ratio either between patients with and without hemoIysis

426

Kattamis and T]ort]atou

16

The Journal o] Pediatrles September 1970

HEMOGLOBIN

_.tSD

meon

I:ll p < 0.2

~----o r o n g e

z_ z rn

12

T

8

I

0 .J 0 0 Ld I

O 20

I

I

1

I

[

I

1

I

RETICULOCYTES 1:.I] p < 0 . 0 5

16 Ul

I.U

~)-

12

o

8

9

= o

; I~1

5O

I

! I~1

I'1

I

BILIRUBIN ]:1] p < 0.01

~' 40 30 z

20 !

__ O CASES GROUPS

9

I

I

9

I I II'l

I

Ia i(-)l(+)

I(-)1(+)

I

Ha

Fig. 3. Mean, standard deviation, and range of lowest hemoglobin and highest reticulocyte and total bilirubin levels in the 5 subgroups of patients studied. (+) ~ hemolysis present; (-) ---~hemolysis absent.

in each of the Groups Ia and Ib, or between normal'and G-6-PD-deficient individuals; (3) correlated reticulocyte counts with bilirubin levels (Fig. 4); no correlation was found in normal children with hemolysis (r = -0.191 or in patients with thalassemia trait (r = +0.42). In contrast, a highly significant correlation (r =.+0.84, p < 0.0011 was noted in patients with G-6-PD deficiency. Red cell osmotic fragility. This parameter was studied in 75 patients by a simple

screening method; the results are shown in Tables III and IV. The osmotic fragility during the initial phase lthird to tenth day) was reduced in 45 (60 per cent), greatly in 34, moderately in 4, and mildly in 7. Between the eleventh to 15th day it was still greatly reduced in 25, restored to normal in 3, and improved in 6. Between the sixteenth to twentieth day it remained greatly decreased in 9 (Table IV). In 4 patients the disturbed osmotic fra-

Volume 77 Number 3

Hemolytic process o[ viral hepatitis

427

171 >~ 151

m11, ILl >(,9

O

7.

_J

w

3 I ~

n1

X

9

J 2

6 10 14 BILIRUBIN

18 IN

22 26 30 m g / 1 0 0 ml

34

38 x

42

Fig. 4. Correlation of total bilirubin levels with reticulocytes in G-6-PD-deficient and G-6PD-nondeficient patients with hemolysis. O ~- G-6-PD deficiency, r - - +0.84; x ~ fl-thalassemia, r ~- +0.42; O : normal hemolysis, r -~ -0.19.

T a b l e I I . R a t i o of u n c o n j u g a t e d : total bilirubin in p a t i e n t s w i t h viral hepatitis GToup$

of uncon]ugated:total bilirubin Ratio

1.0:0.90 0.89:0,80 0.79:0.70 0.69:0.60 0.59:0.50 0,49: 0.40 0.39:0,30 0.29:0.20 0.19:0.10 0.09:0

3 5 7 9 10 5 Total

H (-)

=

la H(-) I H(+)

--

1

.

-

1

.

.

.

.

.

.

H(-) I H(+)

.

1

1

-

10

3

-

3

-

1

-

-

5 2

12 10

8 2

-

1 -

I -

5 I

-

1 -

3

1

-

-

-

2

-

-

37

14

7 (+) =

.

.

H(-) I H(+)

-

-

39

without hemolysis; H

H(-) l H(+)

H(-) [ H(+)

.

4

I

10

1

2

with hemolysis.

g i l i t y r e m a i n e d u n c h a n g e d after r e m o v i n g p l a s m a a n d w a s h i n g the r e d cells with isotonic saline. T h e r a n g e of d e c r e a s e d fragility in t h e a c u t e phase of viral h e p a t i t i s in t h r e e p a tients is ,illustrated i n Fig. 5 , a n d the corr e l a t i o n ~ " decreased :,osmotic fragility to b o t h mean, c o r p u s c u l a r v o l u m e ( M C V ) a n d

m e a n c o r p u s c u l a r h e m o g l o b i n ( M C H ) in Fig. 6. Significant differences in t h e m e a n values of b o t h m e a n c o r p u s c u l a r v o l u m e a n d m e a n c o r p u s c u l a r h e m o g l o b i n were n o t d e t e c t e d b e t w e e n patients w i t h a n d w i t h o u t d i s t u r b a n c e s in fragility, t h o u g h as a rule p a t i e n t s w i t h low m e a n c o r p u s c u l a r v o l u m e a n d m e a n c o r p u s c u l a r h e m o g l o b i n values

4 28

Kattamis and Tjortjatou

The Journal of Pediatrics September 1970

Table IlL Red cell osmotic fragility in 0.36 per cent buffered NaCI solution during the first ten days of illness Red cell osmotic fragility ~ Day of illness

Total cases

No.

3-10

75

30

0 I

%

-[.No.

40

7

1+ I

%

No.

9.2

4

2+ I

%

No.

5.2

34

3+ ] % 45.6

"~0 = normal, 1+ to 3+ ~ decreased fragility.

Table IV. Follow-up of decreased osmotic fragility in 34 patients after the first ten days of illness Days of illness 0-10 11-15 16-20

Red cell osmotic fragility 3+ "l 2+ I 1+ [ 0 34 25 9

4 -

2 2

3 8

had reduced osmotic fragility and both mean values tended to be lower in patients with reduced osmotic fragility. DISCUSSION The clinical course of infectious hepatitis in nondeficlent children and in those with the Mediterranean variant of G-6-PD deficiency B(-) was studied prospectively. It is important to emphasize that this type differs in many respects from the Negro variant, A(-). 11 In the Mediterranean variant young erythrocytes are only partially deficient and male hemizygotes are easily detected even by the brilliant cresyl blue dye test? 2 In this study all deficient males, including those with severe hemolysis, showed no decolorization in > 150 minutes. The brilliant cresyl blue test will not detect more than 50 per cent of female heterozygotes, s Thus it is possible that the slightly higher incidence of hemolysis observed in normal females as compared with males (30:16 per cent) is due to the presence of heterozygotes with mild deficiency who remained undetectable by this test and were considered as normal. Moderate and severe hemolysis developed in deficient patients only. One may speculate on the pathogenesis of severe hemolysis in these patients. It has

been suggested that partially oxidative metabolites which decrease erythrocyte-reduced glutathione are released in infectious hepatitis. Abnormally low levels of glutathione have been demonstrated in the course of the disease, reverting to normal after recovery.13, 14 In G-6-PD deficiency glutathione is already abnormal, as is the capacity of deficient erythrocytes to reduce oxidized glutathione. Further lowering of glutathione in hepatitis impairs the integrity of deficient erythrocytes, leading to their destruction. Hemolysis should be proportional to the degree of involvement of liver function, extensive involvement resulting in the production of large amounts of oxidative metabolites and severe hemolysis, and minor involvement causing mild hemolysis. If this assumption is correct, the pathogenesis of hemolysis in G-6-PD-deficient children with hepatitis is basically similar to that induced by drugs, except that the toxic agents in hepatitis are probably oxidative products arising through liver damage. The extreme bilirubinemia observed in some patients with enzymatic deficiency is difficult to explain. As demonstrated, in normal children bilirubin levels were unaffected by mild hemolysis. On the contrary, a positive correlation was found between the number of reticulocytes and the levels of bilirubin in G-6-PD-deficient children. Furthermore, though the hyperbilirubinemia in four patients was associated with moderate or severe hemolysis, hemolysis was not always associatedwith hyperbilirubinemia, even in G-6-PD-deficient patients. These findings suggest an interrelation between hemolysis and hyperbilirubinemia in G-6PD-deficient children with hepatitis. It

Volume 77 Number 3

Hemolytic process o[ viral hepatitis

100 NORMAL

RANGE

gO

u~

8O

70 W "1-

~ 60 w z 50 U

30

20 QIO

Q20 Q~5 Q30 Q35 OAO OA5 QSO Q55 0~0 0,65 Q70Q75 0,80 Q85 NaCI

%

Fig. 5. Red cell osmotic fragility curves at the initial stage of hepatitis in 3 patients.

9 MCV oMCH

10,5 100

95

35 00 9

0

oee 0 0

90

.=

85

9

80

O0 0

30

9 O0

0

O0

O0 0 O0

M

0 0

oooo M

t--

25

"1" U

U

~;

O0 O0

75

O0

70 65 60

000 0 0000

.E I[

20

Q

SD~7A SD*2.6 o

SD +26,3 SD/d~5

+ OSMOTIC

++

15

++Jr

FRAGILITY

Fig 6. Correlation of red cell fragility with mean corpuscular volume and mean corpuscular hemoglobin.

429

430

Kattamis and Tjortjatou

would not be surprising if in addition to hemolysis other factor or factors, probably of hepatic origin, m a y prove responsible for the great rise of bilirubin in some of the deficient patients; this has already been postulated to explain severe neonatal jaundice. In favor of this hypothesis is the observation that all three G-6-PD-deficient patients with high serum bilirubin levels had had severe neonatM jaundice, for which they had been treated by exchange transfusion and which was unrelated to drug administration or hemolysis. T h e etiology of decreased osmotic fragility, which was present during the initial period of viral hepatitis in most of the patients, is also unknown. Crosby ~s postulated that macrocytes and target cells, which are frequently seen in viral hepatitis, are responsible for lowering osmotic fragility. At least in this study no evidence has been found that either mean corpuscular volume which is related t o macrocytes, or mean red cell hemoglobin content had any significant relation to disturbances of osmotic fragility. Lowering of osmotic red cell fragility is a frequent finding in viral hepatitis; it m a y be difficult to differentiate other conditions with similar disturbances, such as thalassemia trait and iron deficiency. Diagnosis is further confused by the coexistence of mild anemia, hypochromia, target ceI!s, and disturbances of iron metabolism in hepatitis. O n the other hand, the evidence that in heterozygotes with fi-thalassemia, hepatitis frequently caused hemolysis which tended to be rather severe and was sometimes associated with hyperbilirubinemia, points to the necessity of detecting fl-thalassemia before evaluating the clinical course of the disease. Unfortunately, detection of fi-thalassemla trait was not done during the initial phase of this investigation; we believe that among our G-6,PD-nondeficient children with hemolysis were some with thalassemia trait. T h e modification in the spectrum of the clinical course of viral hepatitis in individuals with either G-6-PD deficiency or flthalassemia trait which was demonstrated in

The Journal of Pediatrics September 1970

this study needs careful consideration from clinical, diagnostic, and research aspects, especially in countries in which both hereditary defects are common. REFERENCES 1. Motulsky, A. G.: Theoretical and clinical problems of glucose-6-phosphate dehydrogenase deficiency. Its occurrence in Africans and its combination with hemoglobinopathy; in Jonxis, j. I-I.. P., editor: AbnormaI haemoglobins in Africa, Oxford, 1965, Blackwell Scientific Publications. 2. Szeinberg, A., Sheba, C., Hirshorn, N., and Brodonyi, E. : Studies on erythrocytes in cases of past history of favism and druginduced acute hemolytic anemia, Blood 12: 653, 1957. 3. Burka, E. R., Weaver, Z., and Marks~ P. A.: Clinical spectrum of hemolytic anemia associated with glucose-6-phosphate dehydrogenase deficiency, Ann. Intern. Med. 64: 817, 1966. 4. Salen, G., Goldstein, F., Hanrani, F., and Wilmer, Wirts, G.: Acute hemolytic anemia complicating viral hepatitis in patients with glucose-6-phosphate dehydrogenase deficiency, Ann. Intern. Med. 65- 6, 1966. 5. Choremis, C., Kattamis, C. A., Kyriazakou, M., and Gavriilidou, E.: Viral hepatitis in G-6-PD deficiency, Lancet 1: 269, 1966. 6. Kattamis, C. A., and Kyriazakou, M.: Viral hepatitis and G-6-PD deficiency, Ann. Clin. Paediat. Univ. Athen. 13: 218, 1966. 7. Varley, H.: Practical clinical biochemistry, London, 1963, William & Heinemann (Medical Bks.) Ltd. 8. Kattamis, C. A.: Glucose-6-phosphate dehydrogenase deficiency in female heterozygotes and the X-inactivation hypothesis, Acta Paediat. Scand. Suppl. 172: 103, 1967. 9. Zinkham, W., Lenhard, R. E., and Childs, B.: A deficiency of G-6-PD activity from patients with favism, Bull. Hopkins Hosp. 102: 169, 1958. 10. Parpart, A. K., Lornaz, P. B., Parpart, E. R., Gregg, J. R., and Chase, A. M.: The osmotic resistance (fragility) of human red cells, J. Clin. Invest. 26: 636, I947. 11. WHO: Technical report series. Standardization of procedures for the study of G-6-PD, Geneva, 1967. 12. Kattamis, C. A., Kyriazakou, M., and Chaidas, St.: Favism. Clinical and biochemical data. J. Med. genet. 6: 34, 1969. 13. Jonderko, G.: Glutathione level in the blood m the course of parenchymatous injury of the liver, Arch. Polon. Med. Int. 31: 316, 1961. 14. Pitcher, C. S., and Williams, R.: Reduced red cells survival in jaundice and its relation to abnormal glutathione metabolism, Clin. Scl. 24: 239, 1963. 15. Crosby, W. H.: The pathogenesis of spherocytes and leptocytes (target cells): Analytical review, Blood 1: 261, 1952.