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“Acquired” red cell enzyme defects in hematological diseases
187
CIinica Chimica Acta, 57 (1974) 187-189 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SHORT COMMUNICATION .._ _~__ ____.._ _~~_
_.._ .._. _
_..-.. ._-
CCA 6719
“ACQUIRED” DISEASES
RED CELL ENZYME DEFECTS IN HEMAT~LOGICAL
H. ARNOLDa, K.G. BLWMEa, G.W. LoHRa, M. BOULARDb and Y. NAJEANb ‘Medizinische Ktinik, University of Freiburg, D 78 Freiburg (G.F.R.) Louis, Paris x’ (France)
andb Hiipifaf St.
(Received July 4, 1974)
In a wide variety of different hematological disorders so-called “acquired” enzyme defects of the red blood cell (RBC) have been described. The disorders comprise chronic idiopathic pancytopenia in childhood, aplastic anemia, dyserythropoiesis, refractory anemia, sideroblastic anemia, preleukemia, smoldering leukemia, acute leukemia, polycythemia, and paroxysmal nocturnal hemoglobinuria [l-7]. In most instances pyruvate kinase (PK) was found to be diminished in the patients’ RBC, but other enzymes like glucosephosphate isomerase (GPI), phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GADP), and 2,3-diphosphoglycerate mutase (2,3-DPGM) were found to have decreased activity. In cases of low glutathione reductase (GR), activity has been restored to normal by the in vitro addition of flavin adenine dinucleotide (FAD) in most instances. It must also be mentioned that in some of the dyserythropoietic states with decreased PK activity concomitant increases of other glycolytic enzymes as GPI, GAPD, and 2,3-DPGM and of red cell glutathione have been found which were not related to reticulocytosis. From the observations mentioned the cause-and-effect relationship between enzyme defect and the various diseases seems to be doubtful. To find out about the nature of the “acquired” enzyme defects, RBC from patients with some of the diseases mentioned have been incubated in isologous plasma from healthy donors and in Krebs-Ringer solution containing 5 mM glucose [ 81. After incubation for 4 h at 37” leukocytes and platelets were removed by an earlier described method [9] . After hemolysis [lo] RBC enzyme activities were determined [ 111. The following results have been obtained. With regard to PK activity 6 patients of the Hapital St. Louis, Paris were studied; the results are summarized in Table I. PK activity which was decreased to about half normal values was increased to almost normal activity by incubation in isologous plasma from normal donors. The same observation was made after incubation in KrebsRinger solution. Preincubation of the hemolysates with 0.5 mM 2-mercap-
188
TABLE PK
1
ACTIVITY
MIA
AND
PK
assay
mM
IN
6
PATIENTS
SMOLDERING [12]:
ADP,
100
mM
G U LDH,
’ 1 S.D.,
WITH
SI~ER~BLASTIC
ANEMIA,
CHRONIC
REFRACTORY
ANE-
tEUKEMIA Tris-HCl
1 : 1000
PH
8.0,
hemolysate,
100
mM
KCl,
. Result
37’
10 mM
for normal
MgCI2, samples:
0.2
mM
12.9
NADH. + 2.89
5 mM U PK/e
PEP, Hb
1.5
(mean
II = 10).
Case
PK activity
PK activity
PK activity
(initial
(after
(after
activity)
incubation
m isologous
plasma)
1. Sideroblastic
anemia
7.2
10.8
12.7
2. Sideroblastic
anemia
8.4
11.0
12.3
3. Sideroblastic
anemia
6.9
9.5
9.7
6.3
10.0
11.5
4. Refractory
anemia
5. Smoldering
leukemia
5.0
9.2
9.4
6. Smoldering
leukemia
6.2
10.2
10.5
6.7
10.1
11.0
Mean
value
incubation
with
‘L-ME
toethanol (2-ME) also restored PK activity. RBC-PK from normal donors is not affected by 2-ME (0.1-5.0 mM). In all cases studied PK thermostability [12] was normal. Incubation of patients’ cells in autologous plasma showed no alteration in PK activity. Similar results were obtained with samples from three patients with preleukemia and three patients with acute myelocytic leukemia which were studied in the Medical Clinic of Freiburg. In cross experiments RBC from healthy donors with normal PK activity were incubated in the patients’ plasma, resulting in a significant decrease of enzyme activity. However, this observation was not a universal finding. In a few cases, the inactivating effect of the patients’ plasma on normal cells could not be demonstrated. The reason for this kind of heterogeneity is so far unknown, but it is possible that the inactivating agent cannot be transmitted in all cases. In a case of smoldering leukemia we found a decreased initial PFK activity of 54% of the normal mean. Incubation of the patient’s cells in normal isologous plasma resulted in an increase to 87% of normal PFK activity. When normal RBC were incubated in the patient’s plasma PFK activity dropped to 31%. Experimental data are presented in Table II. Corresponding results were observed for PFK in two patients with aplastic anemia. TABLE
II
PFK-ACTIVITY WITH PFK 1.5
IN
A
SMOLDERING assay
mM
samples:
[ 111:
ATP, 4.1
CROSS-INCUBATION
100
mM
2 U a-GD, t 0.7
EXPERIMENT
Triethanolamine-HCl 2 U TPI,
U PFK/e
Hb
PH
2 U aldolase,
{mean
i S.D.,
_ -...-_-~_~.-..Patient’s
RBC
in autologous
Patient’s
RBC
in normal
Normal
RBC
in autdogous
RBC
in isologous dialyzed
plasma
isologous
Normal
RBC
A
SAMPLE
FROM
A
PATIENT
against
:
7.5, 500
0.5
mM
hemolysate,
EDTA. 2 mM
10 mM F-6-f.
M&12, 25O.
0.2 Result
mM
NADH,
for normal
PFK ._..
activity
2.2 plasma
3.6
plasma plasma
1
n = 10).
Sample
Patient’s
WlTH
LEUKEMIA
4.3 from
buffer
the patient [81 __._____.__
1.2 3.7 ..__
_-.--i_
_^_.
.-
-.... ..--
189
In another case of aplastic anemia we found a diminished GPI activity of 37% which was reversible. Moreover, GR activity was found to be decreased to 43-56s in three cases of acute myelocytic leukemia, one case of aplastic anemia, and in one case of macroglobulinemia. The in-vitro addition of 1 I.~M FAD [ 131 restored GR activity to normal in these non-hereditary cases, as has been described in other reports [3,5,13]. In two other cases with aplastic anemia and decreased GR activity an increase of 20% in enzyme activity was observed after incubation in Krebs-Ringer solution containing glucose [ 81. From the results reported here it can be concluded that the observed decrease in enzyme activity in the diseases mentioned is an unspecific phenomenon which is most probably due to an as yet undefined dialyzable compound of small molecular size. It can be speculated that the inhibiting agent has a sulfhydryl group which forms a mixed disulfide with 2-ME, since preincubation with 2-ME restores enzyme activity to normal. It is unlikely that certain drugs are responsible for the observed phenomenon since some of the patients were studied before they were treated. It is also unlikely that copper is the common denominator as discussed earlier [ 141 ; hexokinase and 6-phosphogluconic dehydrogenase which are most sensitive to copper [14] were not decreased in the conditions mentioned. Our observation reported here does not explain the concomitant increases of some enzyme activities which were mentioned initially. Finally, there is no evidence for a genetic transmission of the biochemical abnormalities discussed. References 1 B. Dreyfus, C. Sultan, H. Rochant. C. Salmon, P. Mannoni, J.P. Cartron, P. Boivin and C. Galand. Brit. J. Haemat., 16 (1969) 303 2 A. Najman, J.P. Leroux, H. Temkine. P. Cartier and R. Andre’, Rev. Franc. Etud. Clin. Biol.. 14 (1969) 795 3 W. SchrGter. Schweiz. Med. Wschr.. 100 (1970) 1101 4 P. Boivin, C. Galand and M. Audolient, Pathol. Biol., 18 (1970) 175 5 U.R. Kleeberg. H. Heimpel, E. Kleihauer und A. OlischlBger, Klin. Wschr.. 49 (1971) 557 6 H. Martin and L. Nowicki, XIV. Gong. Int. Sot. Haemat. Sao Paula 1972, Abstract-Vol. p. 59 7 W.N. Valentine, P.N. Konrad and D.E. Paglia, Blood, 41 (1973) 857 8 H. Arnold, K.G. Blume, R. Engelhardt and G.W. Liihr. Blood, 41 (1973) 691 9 D. Busch and K. Pelz, Klin. Wschr.. 44 (1966) 983 10 G.W. LGhr and H.D. Wailer, Deutsche Med. Wschr.. 86 (1961) 27 11 H. Arnold, K.G. Blume. D. Busch, U. Lenkeit, G.W. Ltihr and E. Liibs, Klin. Wschr., 48 (1970) 1299 12 K.G. Blume, H. Arnold, G.W. LBhr and E. Beutler, Clin. Chim. Acta. 43 (1973) 443 13 E. Beutler. J. Clin. Invest., 48 (1969) 1957 14 M. Boulard, K.G. Blume and E. Beutler, J. Clin. Invest.. 51 (1972) 459
CIinica Chimica Acta, 57 (1974) 187-189 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SHORT COMMUNICATION .._ _~__ ____.._ _~~_
_.._ .._. _
_..-.. ._-
CCA 6719
“ACQUIRED” DISEASES
RED CELL ENZYME DEFECTS IN HEMAT~LOGICAL
H. ARNOLDa, K.G. BLWMEa, G.W. LoHRa, M. BOULARDb and Y. NAJEANb ‘Medizinische Ktinik, University of Freiburg, D 78 Freiburg (G.F.R.) Louis, Paris x’ (France)
andb Hiipifaf St.
(Received July 4, 1974)
In a wide variety of different hematological disorders so-called “acquired” enzyme defects of the red blood cell (RBC) have been described. The disorders comprise chronic idiopathic pancytopenia in childhood, aplastic anemia, dyserythropoiesis, refractory anemia, sideroblastic anemia, preleukemia, smoldering leukemia, acute leukemia, polycythemia, and paroxysmal nocturnal hemoglobinuria [l-7]. In most instances pyruvate kinase (PK) was found to be diminished in the patients’ RBC, but other enzymes like glucosephosphate isomerase (GPI), phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GADP), and 2,3-diphosphoglycerate mutase (2,3-DPGM) were found to have decreased activity. In cases of low glutathione reductase (GR), activity has been restored to normal by the in vitro addition of flavin adenine dinucleotide (FAD) in most instances. It must also be mentioned that in some of the dyserythropoietic states with decreased PK activity concomitant increases of other glycolytic enzymes as GPI, GAPD, and 2,3-DPGM and of red cell glutathione have been found which were not related to reticulocytosis. From the observations mentioned the cause-and-effect relationship between enzyme defect and the various diseases seems to be doubtful. To find out about the nature of the “acquired” enzyme defects, RBC from patients with some of the diseases mentioned have been incubated in isologous plasma from healthy donors and in Krebs-Ringer solution containing 5 mM glucose [ 81. After incubation for 4 h at 37” leukocytes and platelets were removed by an earlier described method [9] . After hemolysis [lo] RBC enzyme activities were determined [ 111. The following results have been obtained. With regard to PK activity 6 patients of the Hapital St. Louis, Paris were studied; the results are summarized in Table I. PK activity which was decreased to about half normal values was increased to almost normal activity by incubation in isologous plasma from normal donors. The same observation was made after incubation in KrebsRinger solution. Preincubation of the hemolysates with 0.5 mM 2-mercap-
188
TABLE PK
1
ACTIVITY
MIA
AND
PK
assay
mM
IN
6
PATIENTS
SMOLDERING [12]:
ADP,
100
mM
G U LDH,
’ 1 S.D.,
WITH
SI~ER~BLASTIC
ANEMIA,
CHRONIC
REFRACTORY
ANE-
tEUKEMIA Tris-HCl
1 : 1000
PH
8.0,
hemolysate,
100
mM
KCl,
. Result
37’
10 mM
for normal
MgCI2, samples:
0.2
mM
12.9
NADH. + 2.89
5 mM U PK/e
PEP, Hb
1.5
(mean
II = 10).
Case
PK activity
PK activity
PK activity
(initial
(after
(after
activity)
incubation
m isologous
plasma)
1. Sideroblastic
anemia
7.2
10.8
12.7
2. Sideroblastic
anemia
8.4
11.0
12.3
3. Sideroblastic
anemia
6.9
9.5
9.7
6.3
10.0
11.5
4. Refractory
anemia
5. Smoldering
leukemia
5.0
9.2
9.4
6. Smoldering
leukemia
6.2
10.2
10.5
6.7
10.1
11.0
Mean
value
incubation
with
‘L-ME
toethanol (2-ME) also restored PK activity. RBC-PK from normal donors is not affected by 2-ME (0.1-5.0 mM). In all cases studied PK thermostability [12] was normal. Incubation of patients’ cells in autologous plasma showed no alteration in PK activity. Similar results were obtained with samples from three patients with preleukemia and three patients with acute myelocytic leukemia which were studied in the Medical Clinic of Freiburg. In cross experiments RBC from healthy donors with normal PK activity were incubated in the patients’ plasma, resulting in a significant decrease of enzyme activity. However, this observation was not a universal finding. In a few cases, the inactivating effect of the patients’ plasma on normal cells could not be demonstrated. The reason for this kind of heterogeneity is so far unknown, but it is possible that the inactivating agent cannot be transmitted in all cases. In a case of smoldering leukemia we found a decreased initial PFK activity of 54% of the normal mean. Incubation of the patient’s cells in normal isologous plasma resulted in an increase to 87% of normal PFK activity. When normal RBC were incubated in the patient’s plasma PFK activity dropped to 31%. Experimental data are presented in Table II. Corresponding results were observed for PFK in two patients with aplastic anemia. TABLE
II
PFK-ACTIVITY WITH PFK 1.5
IN
A
SMOLDERING assay
mM
samples:
[ 111:
ATP, 4.1
CROSS-INCUBATION
100
mM
2 U a-GD, t 0.7
EXPERIMENT
Triethanolamine-HCl 2 U TPI,
U PFK/e
Hb
PH
2 U aldolase,
{mean
i S.D.,
_ -...-_-~_~.-..Patient’s
RBC
in autologous
Patient’s
RBC
in normal
Normal
RBC
in autdogous
RBC
in isologous dialyzed
plasma
isologous
Normal
RBC
A
SAMPLE
FROM
A
PATIENT
against
:
7.5, 500
0.5
mM
hemolysate,
EDTA. 2 mM
10 mM F-6-f.
M&12, 25O.
0.2 Result
mM
NADH,
for normal
PFK ._..
activity
2.2 plasma
3.6
plasma plasma
1
n = 10).
Sample
Patient’s
WlTH
LEUKEMIA
4.3 from
buffer
the patient [81 __._____.__
1.2 3.7 ..__
_-.--i_
_^_.
.-
-.... ..--
189
In another case of aplastic anemia we found a diminished GPI activity of 37% which was reversible. Moreover, GR activity was found to be decreased to 43-56s in three cases of acute myelocytic leukemia, one case of aplastic anemia, and in one case of macroglobulinemia. The in-vitro addition of 1 I.~M FAD [ 131 restored GR activity to normal in these non-hereditary cases, as has been described in other reports [3,5,13]. In two other cases with aplastic anemia and decreased GR activity an increase of 20% in enzyme activity was observed after incubation in Krebs-Ringer solution containing glucose [ 81. From the results reported here it can be concluded that the observed decrease in enzyme activity in the diseases mentioned is an unspecific phenomenon which is most probably due to an as yet undefined dialyzable compound of small molecular size. It can be speculated that the inhibiting agent has a sulfhydryl group which forms a mixed disulfide with 2-ME, since preincubation with 2-ME restores enzyme activity to normal. It is unlikely that certain drugs are responsible for the observed phenomenon since some of the patients were studied before they were treated. It is also unlikely that copper is the common denominator as discussed earlier [ 141 ; hexokinase and 6-phosphogluconic dehydrogenase which are most sensitive to copper [14] were not decreased in the conditions mentioned. Our observation reported here does not explain the concomitant increases of some enzyme activities which were mentioned initially. Finally, there is no evidence for a genetic transmission of the biochemical abnormalities discussed. References 1 B. Dreyfus, C. Sultan, H. Rochant. C. Salmon, P. Mannoni, J.P. Cartron, P. Boivin and C. Galand. Brit. J. Haemat., 16 (1969) 303 2 A. Najman, J.P. Leroux, H. Temkine. P. Cartier and R. Andre’, Rev. Franc. Etud. Clin. Biol.. 14 (1969) 795 3 W. SchrGter. Schweiz. Med. Wschr.. 100 (1970) 1101 4 P. Boivin, C. Galand and M. Audolient, Pathol. Biol., 18 (1970) 175 5 U.R. Kleeberg. H. Heimpel, E. Kleihauer und A. OlischlBger, Klin. Wschr.. 49 (1971) 557 6 H. Martin and L. Nowicki, XIV. Gong. Int. Sot. Haemat. Sao Paula 1972, Abstract-Vol. p. 59 7 W.N. Valentine, P.N. Konrad and D.E. Paglia, Blood, 41 (1973) 857 8 H. Arnold, K.G. Blume, R. Engelhardt and G.W. Liihr. Blood, 41 (1973) 691 9 D. Busch and K. Pelz, Klin. Wschr.. 44 (1966) 983 10 G.W. LGhr and H.D. Wailer, Deutsche Med. Wschr.. 86 (1961) 27 11 H. Arnold, K.G. Blume. D. Busch, U. Lenkeit, G.W. Ltihr and E. Liibs, Klin. Wschr., 48 (1970) 1299 12 K.G. Blume, H. Arnold, G.W. LBhr and E. Beutler, Clin. Chim. Acta. 43 (1973) 443 13 E. Beutler. J. Clin. Invest., 48 (1969) 1957 14 M. Boulard, K.G. Blume and E. Beutler, J. Clin. Invest.. 51 (1972) 459