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A comparative study on the carbohydrate metabolism of glutathione-deficient, glucose-6-phosphate dehydrogenase-deficient and normal red cells
187
SHORT COMMUNICATIONS
BBA 23 223 A comparative study on the carbohydrate metabolism of glutathione-
deficient, glucose-6-phosphate dehydrogenase-deficient and normal human red cells In contrast to normal erythrocytes, the ATP content of human erythrocytes with a deficiency of glucose-6-phosphate dehydrogenase (EC 1.1.1.49 ) gradually decreases after 4 h of incubation at 37 ° in a medium containing glucose and a large amount of one of the oxidation-promoting substances phenylhydrazinO, primaquine 2, or acetylphenylhydrazine3. In the present study it is shown that glutathione-deficient erythrocytes behave similarly in the presence of acetylphenylhydrazine. Furthermore, in an attempt to obtain more information on the metabolism of normal, glutathione-deficient and glucose-6-phosphate dehydrogenase-deficient red cells, chromatographic analysis of red cell extracts before and after incubation with uniformly labelled ~14C~glucose was performed. Glutathione-deficient blood was obtained from 3 patients with hereditary glutathione deficiency4; glucose-6-phosphate dehydrogenase (Glc-6-P-dehydrogenase)deficient blood was obtained from male Caucasians, having Glc-6-P-dehydrogenase deficiency without chronic haemolytic anaemia. The blood was collected by venepuncture and mixed with 1/e volume of a solution containing 2. 7 g of disodium citrate and 2.3 g of glucose/Ioo ml. The mixture was stored or transported at 0-4 ° and was used within 4 h. Erythrocytes were washed 3 times by resuspension in o.15 M NaC1 solution and centrifuged for IO min at room temperature (2000 × g). After each washing the leucocyte layer was sucked off. The resulting packed cells had a haematocrit of approx. 0. 9. Ringer-phosphate solution contained 0.0020 M CaCI 2, 0.0005 M MgC12, O.lO3 M NaC1, 0.0027 M KC1, 0.024 M NaHC03 and 0.0030 M phosphate buffer (pH 7.4). When acetylphenylhydrazine (Schuchardt) and glucose were added, the pH of the final solution had to be readjusted to 7.4. Incubation with acetylphenylhydrazine. 4-ml aliquots of washed packed cells from normal, Glc-6-P-dehydrogenase-deficient and glutathione-deficient subjects were incubated for 8 h at 37 ° with 8-ml portions of a Ringer-phosphate solution containing 316 mg glucose and 750 mg acetylphenylhydrazine/ioo ml. In a blank experiment the acetylphenylhydrazine was omitted. At I-h intervals samples were taken, and ATP 5 and lactate s were determined in the perchloric acid extracts 4. The results are presented in Fig. I. It appears that the lactate production from glucose in all three cell types was not influenced by the presence of acetylphenylhydrazine. The ATP content of the normal erythrocytes was almost constant both in the absence and in the presence of acetylphenylhydrazine. In the glutathione-deficient erythrocytes, however, the ATP content started to decrease after 4 h of incubation with acetylphenylhydrazine, and after 8 h 40-5 ° % of all ATP had disappeared. The erythrocytes of two Glc-6-P-dehydrogenase-deficient subjects behaved in the same way; ATP reduction in the third Glc-6-P-dehydrogenase-deficient sample was much less pronounced, although the Glc-6-P-dehydrogenase activities did not differ significantly (0.5, 0. 4 and 0.5 MARKS units 7, respectively). It is unlikely that the reduction of ATP in glutathione-deficient and Glc-6-P-dehydrogenase-deficient erythrocytes is due to Biochim. Biophys. Acta, 121 (1966) i 8 7 - i 8 9
I~
SHORT COMMUNICATIONS
Lactate
2oL
GSH deficients
/"
20 //
///
10
/ l
0
I
1 2
Gtc- 6 - P dehydrogenose deficients
10
/ r
!
z v//fll
I
3
l
I
4 5
6
I
l
7
8
0
0
1
2
3
4
5
(5 7
8
°o
1
// /,/,
J;J 2 3
4
;%~--
Time (h)
ATP 1.6
1.6
1.6
1.4
1.4
1.4
1.2
1.2
1.2
1.0
1,0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.4
0,2
0.2
0.6
. . . . 0
/ /
/
7//
Z
2°t
ContPols
/
1
2
3
~ , , 4
7
8
0
0.2 1
0
i
~
,
,
i
i
2
3
4
5
6
7
/
B-
O0 T i m e
(h)
Fig. i. L a c t a t e p r o d u c t i o n a n d A T P c o n t e n t in g l u t a t h i o n e - d e f i c i e n t , n o r n l a l a n d G l c - 6 - P - d e h y d r o g e n a s e - d e f i c i e n t r e d cells d u r i n g i n c u b a t i o n in R i n g e r - p h o s p h a t e - g l u c o s e s o l u t i o n w i t h a n d without acetylphenylhydrazine. - ..... , Incubation without acetylphenylhydrazine ;, incub a t i o n w i t h 75 ° m g a c e t y l p h e n y l h y d r a z i n e / I o o m l R i n g e r - p h o s p h a t e - g l u c o s e s o l u t i o n . L a c t a t e a n d A T P a r e e x p r e s s e d a s # m o l e / m l r e d cells.
retardation of glycolysis, effected by acetylphenylhydrazine, as we found that lactate production remained normal in its presence. Erythrocytic ATP is in a dynamic state and therefore the gradual decrease of ATP m a y be caused either by an increased consumption of ATP or by a decreased production of ATP per molecule of lactate during glycolysis in the presence of acetylphenylhydrazine. So far, our investigations gave no indication in either direction. It remains doubtful whether the differences described, found during prolonged incubation in vitro under rather drastic conditions, are correlated with the mechanism of drug-induced haemolysis i~, vivo. Incubation with u~iformly labelled [l*C]glucose. A suspension was made of 4-ml aliquots of normal, glutathione-deficient and Glc-6-P-dehydrogenase-deficient washed packed cells in 8-ml portions of a Ringer-phosphate solution, containing ioo mg glucose/ioo ml. 6 ml of each suspension was used for the preparation of a perchloric acid extract* at zero time; to the remaining 6 ml, Io ~C of uniformly labelled [14C~glucose (specific activity 5 mC/mmole, Philips-Duphar) was added and the resulting suspensions were incubated for 4 h at 37 ° in open test tubes. After the incubation perchloric acid extracts* were prepared. Chromatography of red cell extracts was performed on columns of Dowex-I (formate)S, 9. Tile results of enzymatic and chromatographic analyses of all red cell extracts are summarized in Table I. No significant Biochim. Biophys. Acta, 121 (1966) 1 8 7 - 1 8 9
189
SHORT COMMUNICATIONS TABLE I
SOME METABOLITES OF NORMAL, G L U T A T H I O N E - D E F I C I E N T A N D G L U C O S E - 6 - P H O S P H A T E - D E H Y D R O G E N A S E - D E F I C I E N T ERYTHROCYTES BEFORE A N D AFTER 4 - h OF INCUBATION W I T H UNIFORMLY L A B E L L E D [14C~GLUCOSE IN RINGER--PHOSPHATE SOLUTION AT 37 ° 2,3-Diphosphoglycerate, ATP, ADP, and AMP are expressed as # m o l e / m l cells. Lactate p r o d u c t i o n and glucose c o n s u m p t i o n are expressed as # m o l e / m l cells per 4 h.
Before incubation ATP ADP AMP 2,3-Diphosphoglycerate After incubation ATP ADP AMP 2,3-Diphosphoglycerate Consumed 14C recovered as 2,3-diphosphoglycerate (%) L a c t a t e produced Consumed 14C recovered as lactate (%) Glucose consumed
8 controls
Glutathione deficiency
Glc-6-P-dehydrogenase deficiency
mean
W.-K.
H.K.
Z.-K.
Lu
Ma
P.M.
1.o6 o.12 0.05 2.4
1.12 o.I7 o. I I 2.9
1.o2 o.15 o.09 3.5
I.OO o.19 0.o8 3.4
S.E.
1.13 o.18 0.o6 3.5
o.13 o.o 5 o.o 3 0.9
1.o 5 o.16 0.05 5.5
1.o6 o.I8 0.o6 3.7
1.o 7 o.16 o.o9 1.8
o.o9 o.o 5 o.04 0.5
o.96 o.13 o.o6 2. 4
1.o 5 o.I3 0.o6 2.1
1.I 3 o.I6 o.14 i.o
ii 6. 4
4 1.2
21 6.1
15 6.2
8 4.6
52 3.4
12 1.2
55 2.9
49 3.2
62 2.4
differences were found between the glycolysis of normal, glutathione-deficient and Glc-6-P-dehydrogenase-deficient erythrocytes, being reflected in the amounts of consumed glucose, the yield of labelled metabolites, or the absolute amount of various organic phosphates. Only in the erythrocytes of the glutathione-deficient patient (W.-K.) was the yield of labelled 2,3-diphosphoglycerate comparatively high. So far no evidence has been obtained that the decreased red cell lifespan in glutathione deficiency at normal conditions4 is caused by insufficient protection of red cell carbohydrate metabolism.
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam (The Netherlands)
J. A. Loos C. Z/3RCHER H. K. PRINS
I D. N. MOHLER AND W. J. WILLIAMS, J. Clin. Invest., 4 ° (1961) 1735. 2 G. W. L6HR AND H. D. WALLER, Deut. Med. Wochschr., 86 (1961) 27. 3 J. MAGER, G. GLASER, A. P~AZIN, S. BIEN AND M. 2~'OAM, Biochem. Biophys. Res. Commun., 20 (1965) 235. 4 H. K. PRINS, M. OORT, J. A. Loos, C. ZORCHER AND TH. BECKERS, Blood, 27 (1966) 145. 5 H. ADAM, in H. U. BERGMEYER, Methoden der EmymatischenAnalyse, Verlag Chemie, Weinheim, I962, p. 539. 6 H. J. HOHORST, Biochem. Z., 328 (1957) 509. 7 P. MARKS, Science, 127 (1958) 1338. 8 G. C. MILLS AND L. B. SUMMER, Arch. Biochem., 84 (1959) 79 J. A. Loos, Thesis, University of A m s t e r d a m , 1965, p. 52-
Received December 3rd, 1965 Bioehim. Biophys. Acta, I21 (1966) 187-189
SHORT COMMUNICATIONS
BBA 23 223 A comparative study on the carbohydrate metabolism of glutathione-
deficient, glucose-6-phosphate dehydrogenase-deficient and normal human red cells In contrast to normal erythrocytes, the ATP content of human erythrocytes with a deficiency of glucose-6-phosphate dehydrogenase (EC 1.1.1.49 ) gradually decreases after 4 h of incubation at 37 ° in a medium containing glucose and a large amount of one of the oxidation-promoting substances phenylhydrazinO, primaquine 2, or acetylphenylhydrazine3. In the present study it is shown that glutathione-deficient erythrocytes behave similarly in the presence of acetylphenylhydrazine. Furthermore, in an attempt to obtain more information on the metabolism of normal, glutathione-deficient and glucose-6-phosphate dehydrogenase-deficient red cells, chromatographic analysis of red cell extracts before and after incubation with uniformly labelled ~14C~glucose was performed. Glutathione-deficient blood was obtained from 3 patients with hereditary glutathione deficiency4; glucose-6-phosphate dehydrogenase (Glc-6-P-dehydrogenase)deficient blood was obtained from male Caucasians, having Glc-6-P-dehydrogenase deficiency without chronic haemolytic anaemia. The blood was collected by venepuncture and mixed with 1/e volume of a solution containing 2. 7 g of disodium citrate and 2.3 g of glucose/Ioo ml. The mixture was stored or transported at 0-4 ° and was used within 4 h. Erythrocytes were washed 3 times by resuspension in o.15 M NaC1 solution and centrifuged for IO min at room temperature (2000 × g). After each washing the leucocyte layer was sucked off. The resulting packed cells had a haematocrit of approx. 0. 9. Ringer-phosphate solution contained 0.0020 M CaCI 2, 0.0005 M MgC12, O.lO3 M NaC1, 0.0027 M KC1, 0.024 M NaHC03 and 0.0030 M phosphate buffer (pH 7.4). When acetylphenylhydrazine (Schuchardt) and glucose were added, the pH of the final solution had to be readjusted to 7.4. Incubation with acetylphenylhydrazine. 4-ml aliquots of washed packed cells from normal, Glc-6-P-dehydrogenase-deficient and glutathione-deficient subjects were incubated for 8 h at 37 ° with 8-ml portions of a Ringer-phosphate solution containing 316 mg glucose and 750 mg acetylphenylhydrazine/ioo ml. In a blank experiment the acetylphenylhydrazine was omitted. At I-h intervals samples were taken, and ATP 5 and lactate s were determined in the perchloric acid extracts 4. The results are presented in Fig. I. It appears that the lactate production from glucose in all three cell types was not influenced by the presence of acetylphenylhydrazine. The ATP content of the normal erythrocytes was almost constant both in the absence and in the presence of acetylphenylhydrazine. In the glutathione-deficient erythrocytes, however, the ATP content started to decrease after 4 h of incubation with acetylphenylhydrazine, and after 8 h 40-5 ° % of all ATP had disappeared. The erythrocytes of two Glc-6-P-dehydrogenase-deficient subjects behaved in the same way; ATP reduction in the third Glc-6-P-dehydrogenase-deficient sample was much less pronounced, although the Glc-6-P-dehydrogenase activities did not differ significantly (0.5, 0. 4 and 0.5 MARKS units 7, respectively). It is unlikely that the reduction of ATP in glutathione-deficient and Glc-6-P-dehydrogenase-deficient erythrocytes is due to Biochim. Biophys. Acta, 121 (1966) i 8 7 - i 8 9
I~
SHORT COMMUNICATIONS
Lactate
2oL
GSH deficients
/"
20 //
///
10
/ l
0
I
1 2
Gtc- 6 - P dehydrogenose deficients
10
/ r
!
z v//fll
I
3
l
I
4 5
6
I
l
7
8
0
0
1
2
3
4
5
(5 7
8
°o
1
// /,/,
J;J 2 3
4
;%~--
Time (h)
ATP 1.6
1.6
1.6
1.4
1.4
1.4
1.2
1.2
1.2
1.0
1,0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.4
0,2
0.2
0.6
. . . . 0
/ /
/
7//
Z
2°t
ContPols
/
1
2
3
~ , , 4
7
8
0
0.2 1
0
i
~
,
,
i
i
2
3
4
5
6
7
/
B-
O0 T i m e
(h)
Fig. i. L a c t a t e p r o d u c t i o n a n d A T P c o n t e n t in g l u t a t h i o n e - d e f i c i e n t , n o r n l a l a n d G l c - 6 - P - d e h y d r o g e n a s e - d e f i c i e n t r e d cells d u r i n g i n c u b a t i o n in R i n g e r - p h o s p h a t e - g l u c o s e s o l u t i o n w i t h a n d without acetylphenylhydrazine. - ..... , Incubation without acetylphenylhydrazine ;, incub a t i o n w i t h 75 ° m g a c e t y l p h e n y l h y d r a z i n e / I o o m l R i n g e r - p h o s p h a t e - g l u c o s e s o l u t i o n . L a c t a t e a n d A T P a r e e x p r e s s e d a s # m o l e / m l r e d cells.
retardation of glycolysis, effected by acetylphenylhydrazine, as we found that lactate production remained normal in its presence. Erythrocytic ATP is in a dynamic state and therefore the gradual decrease of ATP m a y be caused either by an increased consumption of ATP or by a decreased production of ATP per molecule of lactate during glycolysis in the presence of acetylphenylhydrazine. So far, our investigations gave no indication in either direction. It remains doubtful whether the differences described, found during prolonged incubation in vitro under rather drastic conditions, are correlated with the mechanism of drug-induced haemolysis i~, vivo. Incubation with u~iformly labelled [l*C]glucose. A suspension was made of 4-ml aliquots of normal, glutathione-deficient and Glc-6-P-dehydrogenase-deficient washed packed cells in 8-ml portions of a Ringer-phosphate solution, containing ioo mg glucose/ioo ml. 6 ml of each suspension was used for the preparation of a perchloric acid extract* at zero time; to the remaining 6 ml, Io ~C of uniformly labelled [14C~glucose (specific activity 5 mC/mmole, Philips-Duphar) was added and the resulting suspensions were incubated for 4 h at 37 ° in open test tubes. After the incubation perchloric acid extracts* were prepared. Chromatography of red cell extracts was performed on columns of Dowex-I (formate)S, 9. Tile results of enzymatic and chromatographic analyses of all red cell extracts are summarized in Table I. No significant Biochim. Biophys. Acta, 121 (1966) 1 8 7 - 1 8 9
189
SHORT COMMUNICATIONS TABLE I
SOME METABOLITES OF NORMAL, G L U T A T H I O N E - D E F I C I E N T A N D G L U C O S E - 6 - P H O S P H A T E - D E H Y D R O G E N A S E - D E F I C I E N T ERYTHROCYTES BEFORE A N D AFTER 4 - h OF INCUBATION W I T H UNIFORMLY L A B E L L E D [14C~GLUCOSE IN RINGER--PHOSPHATE SOLUTION AT 37 ° 2,3-Diphosphoglycerate, ATP, ADP, and AMP are expressed as # m o l e / m l cells. Lactate p r o d u c t i o n and glucose c o n s u m p t i o n are expressed as # m o l e / m l cells per 4 h.
Before incubation ATP ADP AMP 2,3-Diphosphoglycerate After incubation ATP ADP AMP 2,3-Diphosphoglycerate Consumed 14C recovered as 2,3-diphosphoglycerate (%) L a c t a t e produced Consumed 14C recovered as lactate (%) Glucose consumed
8 controls
Glutathione deficiency
Glc-6-P-dehydrogenase deficiency
mean
W.-K.
H.K.
Z.-K.
Lu
Ma
P.M.
1.o6 o.12 0.05 2.4
1.12 o.I7 o. I I 2.9
1.o2 o.15 o.09 3.5
I.OO o.19 0.o8 3.4
S.E.
1.13 o.18 0.o6 3.5
o.13 o.o 5 o.o 3 0.9
1.o 5 o.16 0.05 5.5
1.o6 o.I8 0.o6 3.7
1.o 7 o.16 o.o9 1.8
o.o9 o.o 5 o.04 0.5
o.96 o.13 o.o6 2. 4
1.o 5 o.I3 0.o6 2.1
1.I 3 o.I6 o.14 i.o
ii 6. 4
4 1.2
21 6.1
15 6.2
8 4.6
52 3.4
12 1.2
55 2.9
49 3.2
62 2.4
differences were found between the glycolysis of normal, glutathione-deficient and Glc-6-P-dehydrogenase-deficient erythrocytes, being reflected in the amounts of consumed glucose, the yield of labelled metabolites, or the absolute amount of various organic phosphates. Only in the erythrocytes of the glutathione-deficient patient (W.-K.) was the yield of labelled 2,3-diphosphoglycerate comparatively high. So far no evidence has been obtained that the decreased red cell lifespan in glutathione deficiency at normal conditions4 is caused by insufficient protection of red cell carbohydrate metabolism.
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam (The Netherlands)
J. A. Loos C. Z/3RCHER H. K. PRINS
I D. N. MOHLER AND W. J. WILLIAMS, J. Clin. Invest., 4 ° (1961) 1735. 2 G. W. L6HR AND H. D. WALLER, Deut. Med. Wochschr., 86 (1961) 27. 3 J. MAGER, G. GLASER, A. P~AZIN, S. BIEN AND M. 2~'OAM, Biochem. Biophys. Res. Commun., 20 (1965) 235. 4 H. K. PRINS, M. OORT, J. A. Loos, C. ZORCHER AND TH. BECKERS, Blood, 27 (1966) 145. 5 H. ADAM, in H. U. BERGMEYER, Methoden der EmymatischenAnalyse, Verlag Chemie, Weinheim, I962, p. 539. 6 H. J. HOHORST, Biochem. Z., 328 (1957) 509. 7 P. MARKS, Science, 127 (1958) 1338. 8 G. C. MILLS AND L. B. SUMMER, Arch. Biochem., 84 (1959) 79 J. A. Loos, Thesis, University of A m s t e r d a m , 1965, p. 52-
Received December 3rd, 1965 Bioehim. Biophys. Acta, I21 (1966) 187-189