Red cell survival in galactosemia

T h e J o u r n a l o[ P E D I A T R I C S

619

Red cell survival in galactosemia Erythrocyte survival in a 4~2-year-old galactosemic patient was studied. In vitro autohemolysis o[ the patient's red cells was within normal limits in both the absence and presence of galactose. The hall-lives o[ CrS~-tagged cells in the patient's circulation while he was on a milk-gee diet and in the lather's circulation while he was on an uncontrolled diet were normal. Infusion of a galactose load into the father had no noticeable effect on the survival of the tagged galactosemic red cells. I t is concluded that there are no intrinsic abnormalities in the galactosemic red cell leading to early destruction. It is possible, however, that prolonged exposure to a high serum level o[ galactose may impair red cell survival.

May Y. F. W. Wang,* and Jane F. Desforges** BOSTON~ MASS.

ANEMIA AS A P R O M I N E N T symptom of galactosemia was present in 26 p e r cent of u n t r e a t e d patients in a large series r e p o r t e d by Hsia a n d W a l k e r ? T h e y described the a n e m i a as p a r t l y hemolytic in nature, a n d in vitro incubation of tile p a tient's red cells in a m e d i u m containing galactose revealed increased hemolysis. R e cently we h a d the o p p o r t u n i t y to study autohemolysis a n d r e d ceII survivaI in a child with galactosemia after he h a d been placed on a milk-free diet.

CASE REPORT The treated of 4 ~ mitted

patient, born in Puerto Rico, was first at the Boston City Hospital at the age years for pneumonia. Later he was adto the hospital for further investigation

From the Hematology Laboratory (Tufts), Boston City Hospital, Boston, Mass. Supported by a grant [rom The American Cancer Society (Massachusetts Division). *United States Public Health Service Research Fellow, Hematology Laboratory (TuJts), . Boston City Hospital, Boston, Mass. ~r ProJessor o[ Medicine, Tults University School o[ Medicine, Boston, Mass.

because bilateral cataracts and hepatomegaly had developed. The parents were between 20 and 30 years of age and in good health. Their first child, jaundiced from birth, died in Puerto Rico of an undetermined cause. The development of their second child, our patient, was retarded. Frequent vomiting and attacks of diarrhea afflicted the child from his first month. He was hospitalized seven times in Puerto Rico for treatment. The parents were told that the child was allergic to milk, and the formula was changed many times during infancy. The parents began to observe lenticular opacities and abdominal swelling when the boy was 2 years of age. For the past two years, he had had normal bowel habits and no excessive vomiting. His diet had consisted mainly of juices and cereals. At the time of his admission, the significant physical findings were (1) weight and height less than that of those in the third percentile, (2) bilateral cataracts, and ~3) Prominent abdomen with liver palpable 3 Cm. below the right costal margin, Pertinent laboratory findings were as follows: hematocrit 35 to 37 per cent, negative urinalyses, blood ,sugar 120 mg. per 100

6 2 0

October 1966

W a n g a n d Des[orges

ml., total protein 6.9 Gm. per 100 ml., albumin 2.7 Gm. per 100 ml., serum glutamic oxaloacetic transaminase 123 units, total bilirubin 0.4 mg. per 100 mi., 5 per cent bromsulphalein retention, and 2 plus cephalin flocculation. While in the hospital, the child had occasional guaiac-positive stools presumably due to a small hemorrhoid. Two days after milk was given, galactose was demonstrated in the urine. Milk was then eliminated from the diet. Erythrocyte galaetose-l-phosphate uridyl transferase by the uridine diphosphoglucose consumption method 2 was almost completely absent, confirming the diagnosis of galactosemia. Blood samples from the parents were also examined for this same enzyme; their levels were in the heterozygous range. METHODS

I n vitro erythrocyte autohemolysis was performed by the method of De Gruchy and associates. ~ Sterile defibrinated blood was obtained, and three aliquots were incubated aseptically. T h e first tube contained only blood; the second, blood with glucose to final concentration of 470 rag. per 100 ml.; and the third, blood with galactose to the same concentration. These tubes were placed in a water bath at 37 ~ C. for 48 hours, at which time the percentage of hemolysis was determined. Autologous red cell survival was performed by in vitro tagging of the patient's cells with 15 /ze of Cr 51 as Na2CrO4. T h e first sample for measurement of radioactivity was obtained two hours after intravenous injection of the tagged red cells. Blood samples were then collected at intervals of 1 to 5 days for three weeks, and a final sample was obtained 43 days after the beginning of the study. Radioactivity was measured with a well-type scintillation counter. Counts per milliliter of red cells were plotted against time; from this the half-life of CrS~-tagged red cells were determined. During the entire period of study of red cell survival, daily guaiac tests of the stool were consistently negative. Hemoglobin values remained stable at 11.7 Gm. per 100 ml. with a reticulocyte count ranging from 1 to 1.2 per cent. There was no significant

change in the weight of the patient during this time. T h e effect on erythrocyte survival of short in vivo exposure of galactosemic red cells to a high galactose environment was then studied. T h e patient's cells were tagged with 100 #c of Cr 51 in vitro and injected into the father after prol~er cross-match. The first blood sample was obtained thirty minutes after infusion of the patient's tagged red cells, and serial samples were obtained every 1 to 3 days for the first 12 days of the study. O n the twelfth day, 25 Gin. of galactose ~ was injected intravenously into the father over a period of five minutes. Thereafter, serial blood samples were again obtained from him for a period of 14 days. T h r o u g h o u t the study, tile father's hemoglobin remained stable at 14 Gin. per 100 ml. ; his reticulocyte count was 1.6 to 2 per cent. His diet was unrestricted. T h e data from these two Cr 51 survival studies were fitted independently to an exponential function by the method of least squares. Both fits had a chi-square probability of over 10 per cent, indicating that the in vivo red cell survival tends to follow an exponential function. RESULTS

Results of the in vitro autohemolysis study are presented in Table I, and those of the Cr 51 survival studies are shown in Figs. 1 and 2. I n vitro addition of galactose to a concentration of 470 mg. per 100 ml. had no noticeable effect on the degree of autoCPhanstiehl Laboratories, Inc., Waukegan, Ill.

Table I. I n vitro autohemolysis of galactosemic erythrocytes . Medium

% of hemolysis alter 48 hours of incubation at 37 ~ C. Patient

Blood alone

0.9 per cent

Blood with glucose Blood with galactose

0.5 per cent 0.63 per cent

[

Normal ~

1 to 3.5 per cent 0 to 0.7 per cent

Volume 69 Number 4

R e d cell,survival in galactosemia

621

nificant. T h e slightly shorter half-life of the tagged red cells in the father's circulation cannot be a t t r i b u t e d to exposare to galactose as can be seen from a careful e x a m i n a t i o n of the curve. T h e solid line fit of Fig. 2 shows that the s e r u m level of galactose had no effect on the rate of d i s a p p e a r a n c e of the radioactivity.

I00 90'

,2 70 60

-4 50

,_. 4 0

DISCUSSION

,Io

=~

2'o

zs

31o

315

4'o

DA YS

Fig. 1. Survival of galactosemic patient's red cells in his own circulation. The solid line is a least square fit to an exponential function.

I00 90 k

8o

"~ 70 GALACTOSE

="e~

,~ 50 ~-

4o

'

,'o

,;

.'0

;0

O A YS

Fig. 2. Survival of galactosemic patient's red cells in his father's circulation. The solid line is a least square fit. The dotted Iine is Fig. 1 superimposed. hemolysis. T h e life-span of the patient's celIs in his own circulation was n o r m a l with a half-life of 34 days, while he received a milk-free diet. Moreover, infusion of the patient's cells into the circulation of his father, a heterozygote, d e m o n s t r a t e d a normal survival curve with a half-life of 31 days, while he was on an uncontrolled diet. Because half-lives of C r s~ r e d cell survivals have been r e p o r t e d as 26 days _+ 2 days ~ a n d 33 days _+ 2 days 5 in n o r m a l individuals, the difference of three days in our two Cr sl survival studies does not a p p e a r to be sig-

T h e exact relationship between the absence of g a l a c t o s e - l - p h o s p h a t e uridyl transferase 6 and the clinical manifestations of galactosemia is not known. Exposure to galactose leads to an accumulation of galactose-l-phosphate, 7 which has been shown to inhibit phosphoglucomutase, s h u m a n serum alkaline phosphatase, a n d bovine intestinal phosphatase2 T h e inhibitory action of galact o s e - l - p h o s p h a t e on phosphogtucomutase has been postulated to be responsible for hepatocellular damage, d e v e l o p m e n t of cataracts, and m e n t a l retardation, s Galactose inhibits the growth of the Escherichia coli m u t a n t lacking g a l a c t o s e - l - p h o s p h a t e uridyl transferase b u t not t h a t of the m u t a n t lacking galactokinase. T h e inhibition of g r o w t h of the former strain is associated with an accumulation of g a l a c t o s e - l - p h o s p h a t e in the bacteria. 1~ A c c u m u l a t i o n of g a l a c t o s e - l - p h o s p h a t e has been suggested to interfere with e r y t h r o cyte oxygen consumption 7 a n d A T P synthesis a n d m a i n t e n a n c e ? ~ Studies p e r f o r m e d on the r e d cells of one g a i a c t o s e m i c child by Penington and P r a n k e r d d e m o n s t r a t e d a fall in e r y t h r o c y t e adenosine t r i p h o s p h a t e ( A T P ) a n d d i p h o s p h o g l y c e r a t e ( D P G ) content while the child was on a n o r m a l diet. These p h o s p h a t e esters r e t u r n e d to n o r m a l levels when milk was eliminated from the d i e t ? ~ I m p a i r e d m a i n t e n a n c e of a d e q u a t e A T P levels leads to a decIine in active transp o r t a n d p r e s u m a b l y to a decrease in the viability of the red cells. 12 I n fact, B a a r and G o r d 0 n 9 have d e m o n s t r a t e d an i n c r e a s e in p o t a s s i u m efflux reflecting i m p a i r m e n t of active t r a n s p o r t when galactosemic red cells were i n c u b a t e d with galactose. 'By inference, then, a c c u m u l a t i o n of g a l a c t o s e - l - p h o s p h a t e

622

Wang and Des[orges

m a y interfere with red cell sutMval in a galactosemic patient. O n the other hand, recent studies by Zipursky a n d associates la d e m o n s t r a t e d t h a t the turnover of A T P a n d 2,3 D P G in erythrocytes of galactosemic subjects was not i m p a i r e d after one h o u r of incubation in the presence of galactose in spite of the accumulation of galactose-l-phosphate. O u r autohemolysis study revealed no increase in hemolysis after i n c u b a t i o n of galactosemic cells with galactose for 48 hours. W e did not d e t e r m i n e the level of g a l a c t o s e - l - p h o s p h a t e at the end of this incubation period, b u t we can justifiably assume f r o m the work of others that the erythrocytes did a c c u m u l a t e galactose-l-phosphateY, la I t appears, therefore, t h a t in vitro exposure of galactosemic red cells to increased levels of galactose-1p h o s p a t e for as long as two days does not lead to increased hemolysis. O u r in vivo C r 51 r e d cell survival studies showed n o r m a l life-span of the galactosemic erythrocytes in both the absence a n d presence of galactose. I n t r a v e n o u s a d m i n i s t r a t i o n of 25 Gm. of galactose to n o r m a l adults results in measurable amounts of galactose in the serum one h o u r after injection, and about half of those heterozygous for galactosemia show m o r e p r o l o n g e d elevation of serum galactose t h a n n o r m a l subjects after a galactose l o a d ? 4, 1~, 16 I n vivo exposure of galactosemic red cells to galactose for as short as half an h o u r can lead to intraerythrocytic a c c u m u l a t i o n of galactose-1p h o s p h a t e ? 5 W e d i d not measure galactose or g a l a c t o s e - l - p h o s p h a t e levels in o u r in vivo study. However, we can assume t h a t there was at least transient a c c u m u l a t i o n of galact o s e - l - p h o s p h a t e in the galactosemic red cells when the galactose l o a d was administered. Thus, we conclude t h a t there is no intrinsic a b n o r m a l i t y in the red cell struct u r e of galactosemic patients which leads to p r e m a t u r e destruction of red cells b u t r a t h e r t h a t the hemolytic states in u n t r e a t e d p a tients 1 a n d the in vitro m e t a b o l i c defects 7, 9, 11 are due to an adverse environment. O u r studies also d e m o n s t r a t e t h a t limited contact with galactose or galactose-

October 1966

1-phosphate does not interfere with viability of galactosemic red cells. Consequently it is assumed t h a t such a c q u i r e d d e f e c t s are observed only after exposure for a more p r o l o n g e d time or to higher concentrations or to both. SUMMARY

1. I n vitro incubation of r e d cells from a galactosemic subject w i t h galactose for 48 hours at 37 ~ C. did n o t lead to any increase in hemolysis. 2. Erythrocyte life-span was n o r m a l in a galactosemic p a t i e n t on a milk-free diet. 3. T h e red cells of the galactosemic patient h a d a n o r m a l life-span after transfusion into his heterozygous father, who was on an unrestricted diet. 4. Short term in vivo exposure of galactosemic red cells to a galactose load did not i m p a i r their survival. REFERENCES

1. Hsia, D. Y.-Y., and Walker, F. A.: Variability in the clinical manifestations of galactosemia, J. PEDIAT. 59: 872, 1961. 2. Sigma Technical Bulletin, No. 600--UV, Revised, St. Louis, Mo., November, 1963. 3. De Gruchy, G. C., Santamaria, J. M., Parsons, I. C., a~d Crawford, H.: Nonspherocytic congenital hemolytic anemia, Blood 16: 1371, 1960. 4. Lewis, S. M., Szur, L., and Dacie, J. V.: The pattern of erythroeyte destruction in hemolytic anemia as studied with radioactive chromium, Brit. J. Ilaemat. 6: 122, 1960. 5. Jandl, J. H., Greenberg, M. S., Yonemoto, R. H., and Castle, W. B.: Clinical determination of the sites of red cell sequestration in hemolytic anemias, J. Clin. Ir~vest. 35: 842, 1956. 6. Kalckar, H. M., Anderson, E. P., and Isselbacher, D. J.: Galactosemia, a congenital defect in a nucleotide transferase, Biochem. et biophys, acta 20: 262, 1956. 7. Schwarz, V., Goldberg, L., Komrower, G. M., and Holzel, A.: Some disturbances of erythrocyte metabolism in galactosemla, Biochem. J. 62: 34, 1956. 8. Sidbury, J. B., Jr.: The role of galactose1-phosphate in the pathogenesis of galactosemia, in Gardner, L. I., editor: Molecular genetics and human disease, Springfield, Ill., 1961, Charles C Thomas, Publisher. 9. Baar, H. S., and Gordon, M.: Cation-fluxes in galactosemic erythrocytes, Nature 201: 1223, 1964. 10. Durahashl, K., and Wahba, A. J.: Interference with growth of certain Escherichia

Volume 69

Number 4

coli mutants by galactose, Biochem. et biophys. acta 30: 298, 1958. 11. Penington, J. S., and Prankerd, T. A. J.: Studies of erythrocyte phosphate ester metabolism in galactosemia, Clin. Sc. 17: 385, 1958. 12. Jandl, J. H.: Leaky red cells, Blood 26: 367, 1965. 13. Zipursky, A., Rowland, M., Ford, J. D., Haworth, J. C., and Israels, L. G.: Erythro-

R e d cell survival in galactosemia

623

cyte metabolism in galactosemia , Pediatrics 35: 126, 1965. 14. King, E. J., and Aitken, R. S.: An intravenous galactose tolerance test, Lancet 2: 543, 1940. t5. Donnell, G. N., Bergren, W. R., Perry, G., and Koch, R.: Galactose-l-phosphate in galactosemia, Pediatrics 31: 802, 1963. 16. Holzel, A., and Komrower, G. M.: A study of the genetics of galactosemia, Arch. Dis. Childhood 30: 115, 1965.