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An improved procedure for the diagnosis of chronic granulomatous disease, using concanavalin A and cytochalasin E
173
Clinica Chimica Acta, 0 Elsevier/North-Holland
74 (1977) 173-176 Biomedical Press
SHORT COMMUNICATION CCA
8185
AN IMPROVED PROCEDURE FOR THE DIAGNOSIS OF CHRONIC GRANULOMATOUS DISEASE, USING CONCANAVALIN A AND CYTOCHALASIN E
AKIRA
NAKAGAWARA
Department (Received
*, B.Z.
of Biochemistry, June
25th,
FERDAIS
Kyushu
NABI
University
** and School
SHIGEKI
MINAKAMI
of illedicine,
Fuhuoha
812
(Japan)
1976)
Introduction We have previously sh,own that cytochalasin E (Cyto E) induces in human polymorphonuclear leucocytes (PMN) the release of superoxide anions (0;) and that the reaction can be used for a diagnostic test of chronic granulomatous disease (CGD) [ 11. Later observation that the treatment of PMN with concanavalin A (Con A) intensifies the reaction, together with the finding that PMN prepared by hypotonic treatment of blood can be used for the test, led us to propose a new simple diagnostic test of CGD which needs only a small amount of blood. Method A blood sample (0.5 ml) was pipetted into ice-cold isotonic saline solution buffered with 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, pH 7.4 (HEPES-saline). The suspension was hemolyzed by the addition of ice cold water (10 ml), kept in an ice bucket for 50 set and made isotonic by the addition of 10 ml 300 mM NaCl solution. The suspension was centrifuged at 300 X g for 7 min in a refrigerated centrifuge. The precipitate was suspended in 10 ml HEPES-saline and centrifuged at 300 X g for 7 min, and the resulting pellet was suspended in 0.45 ml HEPES-saline and kept in an ice bucket until use. The number of PMN in the final suspension was about 4 X 106/ml. The PMN suspension was added to 1.0 ml HEPES-saline containing 2 mM glucose, 65 PM ferricytochrome c and 1.0 mM CaCl, and preincubated in a
* To * * On
whom leave
correspondence from
the
Institute
should
he
addressed.
of Nutrition,
University
of Dacca,
Dacca-2.
Bangladesh.
174
plastic spectrophotometric cuvette at 37°C for 8 min. The cuvette was put in a thermostatted cuvette holder (37°C) of a dual wave-length spectrophotometer * (Hitachi 556) and the reduction of cytochrome c was measured at 550 nm with a reference wavelength at 540 nm, with the full scale of the recorder set at 0.1 absorbance. 5 ~1 each of Con A, Cyto E and superoxide dismutase (SOD) were sequentially added to the cuvette using a glass rod, while the time course of the cytochrome reduction was followed on the recorder. Con A was added first and after 7 min Cyto E was added. SOD was finally added to ascertain the reduction due to the release of 0;. Concanavalin A (Con A) (Sigma Chem. Co.) was dissolved in isotonic saline solution (10 mg/ml) and cytochalasin E (Cyto E) (Aldrich Chem. Co.) was dissolved in dimethylsulfoxide (2.0 mg/ml). They can be kept in a freezer for at least 2 months. Stock solution of Cyto E was diluted 1 : 1 with HEPES-saline immediately before use. The final concentration of dimethylsulfoxide in the cuvettes was 2.5 pi/ml which does not appreciably interfere the 0, release. Results Figs. 1 and action of PMN centration 50 quent addition
2a show a typical spectrophotometric trace of the 0, from normal subjects. The addition of Con A to PMN pg/ml) induces some but usually a negligible release of of Cyto E (final concentration 5 ,ug/ml) ** induced the
release re(final con0;. Subsereduction
SOD
( 105cells
PMNs
Fig.
1. Reduction
canavalin Cvto
A
of
and
exogenous
cytochalasin
E as a function
* A split-beam result.
**
Con
of cell
then
cytochrome E.
The
such
c by
human
insert
shows
with
side-on
the
it is advisable as a Hitachi
to 556
use
Cyto
of
0;
release
or
photomultiplier
a double
Aminco
E can
also
be added
wavelength
DW-2,
be neglected.
A and
polymorphonuclear rate
laucocytes at 6 min
treated after
the
with
con-
addition
of
numbers.
spectrophotometcr
However.
multiplier can
)
simultaneously.
since
(Hitachi
124)
gave
spectrophotometer the
scattering
of light
essentially with by
the same
end-on suspended
photoPMN
Con. A
Control
Patient
Cyt0.E
SOD i
of CGD
Fig. 2. Cytochrome c reduction by leucocytes of a normal tnus disease. The number of PMN cells was 1.5 X 105.
subject and a patient
with chronic
granulon~a-
of cytochrome c, this being completely inhibited by the addition of SOD (final concentration 10 pg/ml). The rate of cytochrome c reduction was more than 50 times greater compared with the experiment without Con A pretreatment. Although the cytochrome reduction was actually not linear with time, we may use an apparently linear portion for the assay. The insert in Fig. 1 shows the relationship between the number of PMN used and the rate of cytochrome c reduction, which is essentially proportional. The rate of cytochrome reduction was 0.69 t 0.02 (S.E.M., y1= 5) nmol/min per lo5 cells at 6 min after the addition of Cyto E. When PMN of a CGD patient (female, 8 years [ 11) was tested with this procedure, the rate of cytochrome c reduction was less than 2% of the normal value (Fig. 2b). The PMN fraction separated by the Conray-Ficoll method [2] showed more than 90% of the 0; release activity of blood and essentially no reaction was found in red cells and in platelets. The lymphocyte fraction showed some activity, probably due to the monocytes present. Discussion There are several advantages to this test over the nitrobluetetrazolium (NBT) test [3] which is currently in use. The numbers of PMN necessary for the present test was 1 X 10’ cells whereas the NBT test uses 2.5 X lo6 cells for a single incubation. Moreover, the reduction of cytochrome c can be followed continuously in a single sample with sequential additions of Cyto E and SOD, so that control experiments with separate samples are not necessary. The test is apparently specific for bactericidal activity of PMN: essentially no reaction was observed for PMN from a CGD patient. The present test is possible to carry out routinely for the diagnosis of CGD with a minute amount of blood (0.1 ml or less) even from a new born and may also be helpful to detect the CGD carrier state of mothers. Acknowledgement We thank Dr. S. Miyazaki of Department of Pediatrics, Kyushu University Hospital and Dr. K. Motomura of the Fukuoka Red Cross Hospital for their
176
help in taking the blood sample of the CGD patient. This study was supported in part by the Yamanouchi Foundation of Metabolism and Diseases and a Research Grant from the Ministry of Education (Japan). References 1
Nakagawara.
A.,
Kakinuma,
K.,
Shin.
H..
Miyuaki.
S.
and
Minakami,
133-137 2
B&urn,
3
Baehner,
A.
(1968)
R.I.
and
Stand. Nathan,
J. Clin. D.G.
Lab.
(1968)
Invest. New
21, En%
Suppl. J. Med.
97 278,
971-976
S.
(1976)
Clin.
Chim.
Acta
70.
Clinica Chimica Acta, 0 Elsevier/North-Holland
74 (1977) 173-176 Biomedical Press
SHORT COMMUNICATION CCA
8185
AN IMPROVED PROCEDURE FOR THE DIAGNOSIS OF CHRONIC GRANULOMATOUS DISEASE, USING CONCANAVALIN A AND CYTOCHALASIN E
AKIRA
NAKAGAWARA
Department (Received
*, B.Z.
of Biochemistry, June
25th,
FERDAIS
Kyushu
NABI
University
** and School
SHIGEKI
MINAKAMI
of illedicine,
Fuhuoha
812
(Japan)
1976)
Introduction We have previously sh,own that cytochalasin E (Cyto E) induces in human polymorphonuclear leucocytes (PMN) the release of superoxide anions (0;) and that the reaction can be used for a diagnostic test of chronic granulomatous disease (CGD) [ 11. Later observation that the treatment of PMN with concanavalin A (Con A) intensifies the reaction, together with the finding that PMN prepared by hypotonic treatment of blood can be used for the test, led us to propose a new simple diagnostic test of CGD which needs only a small amount of blood. Method A blood sample (0.5 ml) was pipetted into ice-cold isotonic saline solution buffered with 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, pH 7.4 (HEPES-saline). The suspension was hemolyzed by the addition of ice cold water (10 ml), kept in an ice bucket for 50 set and made isotonic by the addition of 10 ml 300 mM NaCl solution. The suspension was centrifuged at 300 X g for 7 min in a refrigerated centrifuge. The precipitate was suspended in 10 ml HEPES-saline and centrifuged at 300 X g for 7 min, and the resulting pellet was suspended in 0.45 ml HEPES-saline and kept in an ice bucket until use. The number of PMN in the final suspension was about 4 X 106/ml. The PMN suspension was added to 1.0 ml HEPES-saline containing 2 mM glucose, 65 PM ferricytochrome c and 1.0 mM CaCl, and preincubated in a
* To * * On
whom leave
correspondence from
the
Institute
should
he
addressed.
of Nutrition,
University
of Dacca,
Dacca-2.
Bangladesh.
174
plastic spectrophotometric cuvette at 37°C for 8 min. The cuvette was put in a thermostatted cuvette holder (37°C) of a dual wave-length spectrophotometer * (Hitachi 556) and the reduction of cytochrome c was measured at 550 nm with a reference wavelength at 540 nm, with the full scale of the recorder set at 0.1 absorbance. 5 ~1 each of Con A, Cyto E and superoxide dismutase (SOD) were sequentially added to the cuvette using a glass rod, while the time course of the cytochrome reduction was followed on the recorder. Con A was added first and after 7 min Cyto E was added. SOD was finally added to ascertain the reduction due to the release of 0;. Concanavalin A (Con A) (Sigma Chem. Co.) was dissolved in isotonic saline solution (10 mg/ml) and cytochalasin E (Cyto E) (Aldrich Chem. Co.) was dissolved in dimethylsulfoxide (2.0 mg/ml). They can be kept in a freezer for at least 2 months. Stock solution of Cyto E was diluted 1 : 1 with HEPES-saline immediately before use. The final concentration of dimethylsulfoxide in the cuvettes was 2.5 pi/ml which does not appreciably interfere the 0, release. Results Figs. 1 and action of PMN centration 50 quent addition
2a show a typical spectrophotometric trace of the 0, from normal subjects. The addition of Con A to PMN pg/ml) induces some but usually a negligible release of of Cyto E (final concentration 5 ,ug/ml) ** induced the
release re(final con0;. Subsereduction
SOD
( 105cells
PMNs
Fig.
1. Reduction
canavalin Cvto
A
of
and
exogenous
cytochalasin
E as a function
* A split-beam result.
**
Con
of cell
then
cytochrome E.
The
such
c by
human
insert
shows
with
side-on
the
it is advisable as a Hitachi
to 556
use
Cyto
of
0;
release
or
photomultiplier
a double
Aminco
E can
also
be added
wavelength
DW-2,
be neglected.
A and
polymorphonuclear rate
laucocytes at 6 min
treated after
the
with
con-
addition
of
numbers.
spectrophotometcr
However.
multiplier can
)
simultaneously.
since
(Hitachi
124)
gave
spectrophotometer the
scattering
of light
essentially with by
the same
end-on suspended
photoPMN
Con. A
Control
Patient
Cyt0.E
SOD i
of CGD
Fig. 2. Cytochrome c reduction by leucocytes of a normal tnus disease. The number of PMN cells was 1.5 X 105.
subject and a patient
with chronic
granulon~a-
of cytochrome c, this being completely inhibited by the addition of SOD (final concentration 10 pg/ml). The rate of cytochrome c reduction was more than 50 times greater compared with the experiment without Con A pretreatment. Although the cytochrome reduction was actually not linear with time, we may use an apparently linear portion for the assay. The insert in Fig. 1 shows the relationship between the number of PMN used and the rate of cytochrome c reduction, which is essentially proportional. The rate of cytochrome reduction was 0.69 t 0.02 (S.E.M., y1= 5) nmol/min per lo5 cells at 6 min after the addition of Cyto E. When PMN of a CGD patient (female, 8 years [ 11) was tested with this procedure, the rate of cytochrome c reduction was less than 2% of the normal value (Fig. 2b). The PMN fraction separated by the Conray-Ficoll method [2] showed more than 90% of the 0; release activity of blood and essentially no reaction was found in red cells and in platelets. The lymphocyte fraction showed some activity, probably due to the monocytes present. Discussion There are several advantages to this test over the nitrobluetetrazolium (NBT) test [3] which is currently in use. The numbers of PMN necessary for the present test was 1 X 10’ cells whereas the NBT test uses 2.5 X lo6 cells for a single incubation. Moreover, the reduction of cytochrome c can be followed continuously in a single sample with sequential additions of Cyto E and SOD, so that control experiments with separate samples are not necessary. The test is apparently specific for bactericidal activity of PMN: essentially no reaction was observed for PMN from a CGD patient. The present test is possible to carry out routinely for the diagnosis of CGD with a minute amount of blood (0.1 ml or less) even from a new born and may also be helpful to detect the CGD carrier state of mothers. Acknowledgement We thank Dr. S. Miyazaki of Department of Pediatrics, Kyushu University Hospital and Dr. K. Motomura of the Fukuoka Red Cross Hospital for their
176
help in taking the blood sample of the CGD patient. This study was supported in part by the Yamanouchi Foundation of Metabolism and Diseases and a Research Grant from the Ministry of Education (Japan). References 1
Nakagawara.
A.,
Kakinuma,
K.,
Shin.
H..
Miyuaki.
S.
and
Minakami,
133-137 2
B&urn,
3
Baehner,
A.
(1968)
R.I.
and
Stand. Nathan,
J. Clin. D.G.
Lab.
(1968)
Invest. New
21, En%
Suppl. J. Med.
97 278,
971-976
S.
(1976)
Clin.
Chim.
Acta
70.