- Email: [email protected]
A radioimmunoassay of human circulatory dopamine-β-hydroxylase
Life Sciences Vol . 13, pp. 769-774, 1973 . Printed in Great Britain
Pergamon Press
A RADIOIMMUNOASSAY OF HUMAN CIRCULATORY DOPAMINE-ß-HYDFWXYLASIE Richard P. Ebstein,
Dong H. Park, Lewis S. Freedman, Stuart M. Levitz,
Takeshi Ohuchi and Menek Goldstein New York University Medical Center, Dep artment of Psychiatry Neurochemistry Laboratories, New York, N.Y. 10016
(Received 1 August 1973 ; in final form 21 August 1973) summary A procedure for the radioimmunoassay of human circulatory dopamine-ß-hydroxylase (DßH) using antibodies directed toward human DpH is described. A significant correlation between serum DßH ac tivity and the amount of serum immunoreactive (MR) DßH was found in the analyzed population . The radioimmunoassay of serum DßH could be used in monitoring changes of sympathetic activity in various physiological and pathological states . Introduction The levels of serum dopamine-ß-hydroxylass
(EC 1.14.2 .1) activity vary
widely in the normal population but are maintained at a relatively constant level in each individual
(1,2) .
The DßH activity in the serum may not reflect the
amount of the enzyme released from synaptic vesicles following sympathetic stimulation since the enzyme might be inactivated to some extent prior to its removal from the circulation.
The radioimmunoassay can be applied to the estimation of
the total IR enzyme-protein
(active and inactive enzyme) and this procedure is
not dependent on the labile enzyme activity.
Recently a solid-radioimmunoassay
utilizing antibodies to sheep adrenal DßH and 1 was described (3) .
125
labelled sheep adrenal DßH
Since there is a considerable cross species loss in i mn,n o-
reactivity (4) we have modified the radioimmunoassay for determination of circulatory human DßH by. using 1
125
labelled human DßH to inhibit competitively the
binding of human serum DßH to antibody directed toward human enzyme .
In this
communication we will describe the procedure for the determination of human serum 1 This work was supported by PHs Grant MH02717 and NSF Grant GB27603.
769
770
Assay for Dopamine-ß-Hydroxylase
Vol . 13, No. 7
DßH by the radioimmunoassay and we will compare the total serum
Ijpft
protein lev-
els with the serum DßH activity levels in humans . Methods DßH was purified from a human pheochromocytoma tumor .
The tumor was ob-
tained immediately after surgery and the enzyme was purified from the 100,000 x g particulate fraction by the previously described procedure (5) .
As a final puri
fication step the enzyme preparation was submitted to a linear sucrose density gradient centrifugation (Park,
Ebstein and Goldstein,
unpublished data) .
The
electrophoresic analysis of the final preparation after sucrose density gradient centrifugation showed only a single band of DßH.
Human antibodies against puri-
fied human DßH were produced in rabbits and tested for specificity by imm noelectrophoresis .
Following immunoelectrophoresis human antiserum gives a single
precipitin line with DßH preparations obtained from human pheochromocytomas but does not give a precipitin line with DßH preparations obtained from bovine adrenal glands . al,
(6),
DßH was iodinated for use as a tracer by the method of Greenwood et
To a solution of 25 Wg purified DßH in 0,5 M phosphate buffer at pH 7,5
was added 2 me of sodium iodide-125
(sp. act,
T in 10 ~L1 of phosphate buffer saline (PBS) . sec.
17 Ci/mg)
and 0.1 mg of chloramine-
The reaction was stopped after 30
by the addition of 0.2 mg sodium metabisulfite in 200 ~L1 of PBS and 2 mg of
KI in 0.2 ml was added.
The solution was passed through a column (1 x 22 cm) of
Sephadex G-25 equilibrated with PBS,
and the eluted iodine labelled protein was
then submitted to a linear sucrose density gradient (5-208 sucrose) . tion containing 1
125
labelled DßH was used for the radioimmunoassay .
The fracThe serum
DßH levels were measured by the solid-state radioimmunoassay as described by Catt and Tregear (7) .
1
125
labelled DßH was incubated in antibody-coated tubes
for 48 hrs with either standard amounts of purified DßH or serum samples. rinsing with water,
After
the dry tubes were counted in a well counter (Nuclear Chicago,
1185 Automatic Gamma Counter ),
DßH activity in the serum was determined by the
previously described procedure (8),
Vol. 13, No. 7
Assay for Dopamine-ß-Hydroxylase Fig.
1.
9
7
a u 5
4
3
1 1 . 0
10
.
20 rig DOFI
.
30
40
standard radioiauunoassay curve for . human DPI Fig.
Z.
50r-
40
É
30
10
0
20
40 60 80 Enzymatic activity (units)
100
The relationship between serum DßH activity and serum üt DßH in humans. The correlation coefficient - 0 .80 P < .01 (N-60)
771
77 2
Vol. 13, No . 7
Assay for Dopamine -ß-Hydroxylase TABLE 1 The Relationship of Human Serum DßH Activity to Immunoreactant DßH Levels
No. of Subjécts *
IR DßH ***
DßH Activity** (range)
4g/ml
19
0-20
6.1
± 0.69
21
21-40
11.6
± 1.09
20
41-90
20 .49 ± 1.67
There was no significant difference between the analyzed normal children (age group S-16) and analyzed normal adults in the mean values of the serum IR DßH amounts and in the serum DßH activities . One unit is defined as the enzyme activity that foams 1 nmole of C14-N-methylated product per ml of serum per 20 min incubation time . ***
These results represent the means ± S.E .M.
Results Figure 1 shows the standard radioincwso assay curve for human DPI, tion of 1
125
labelled DßH was linear over the range 1-20 ng DPIL
Inhibi
The standard
error of the mean after four determinations on a single sample was 5% of the mean assay.
Several dilutions of human serum and standard preparations of purified
human DßH showed parallel decreases in inhibition.
The total circulatory IR an-
zyme-protein levels and the enzyme activity levels were analyzed in 35 normal individuals and in 25 individuals with various mental and neurological disorders of both sexes and various age groups .
Serum IR enzyme-protein levels vary among the
analyzed individuals but the range of values is narrower than that of the enzymatic activities .
There is a preponderance of sera with low IR enzyme-protein
levels in the low enzyme activity group and a preponderance of sera with high IR enzyme-protein levels in the high enzyme activity group.
The differences in the
mean values of serum IR enzyme-protein levels between different enzyme activity
Assay for Dopamine -ß- Hydroxylase
Vol . 13, No. 7
groups is statistically significant (Table 1) .
773
A plot of enzyme activity levels
versus IR enzyme-protein levels reveals that there is a significant correlation between enzyme activity levels and IR enzyme-protein levels (Fig .
2) .
When the
enzyme activity levels versus IR enzyme-protein levels were separately plotted for the normal population and for the population consisting of mental and neurological patients similar correlation coefficients were obtained . Discussion The present study describes a radio immun oassay for determination of human circulatory DßH.
125 This assay employs 1 labelled human DßH and antibodies di-
rected toward human DßH, while the previously described assay (3) employs 1 labelled sheep DßH and antibodies directed toward sheep DßH.
125
Thus, the lower
values obtained for human serum IR DßH by the previously described radioimmunoassay (3) as compared with the presently described assay might be due to a loss of cross-immunoreactivity between species.
It is of interest to note that with
the sheep radioimmunoassay system no correlation between human serum IR enzymeprotein levels and enzyme activity was found (9) .
In this study we have shown a
significant correlation between human serum IR enzyme-protein levels and enzyme activity levels.
These findings indicate that the radioimmunoassay employed in
this study reflects the DßH levels in human serum.
Furthermore,
preliminary
studies show that the radioimmunoassay is reliable in detecting acute changes in sympathetic activity . The simultaneous determination of circulatory DßH activity and of total enzyme-protein levels could be advantageous .
A correlation,
or lack of correlation
between these two assays will indicate whether the alterations in circulatory en zyme levels are due to changes in the rate of enzyme release or to changes in the rate of enzyme inactivation prior to its elimination from the circulation.
In a
separate communication we will report on the circulatory human IR DßH levels and on enzyme activity levels in various physiological and pathological states .
774
Assay for Dopamine -ß- Hydroxylase
Vol . 13, No. 7
References 1.
J. F. LINGLEY, R H, SAGERMAN, 277, 1227-1230 (1967) .
2.
R M. WEINSHILBOUM and J. AXELROD, N. Eng. J. Med. 285,
3.
R. A. RUSH and L. H. GEFFEN, Cir. Res. 31, 444-452 (1972) .
4.
T. OHUCHI, T. H. JOH, L. S. FREEDMAN and M. GOLDSTEIN, on Pharmacol. Abstr. 1022, p. 171 (1972) .
5.
M. GOLDSTEIN, in : Methods in Neurochemistry, Chapter 13, pp. N. Marks and R Rodnight. Plenum Press, New York (1972) .
6.
F. C. GREENWOOD, (1963) .
7.
K. J. GATT and G. W. TREGEAR,
8.
M. GOLDSTEIN,
9.
R. A. RUSH, T. NAGATSU, Abstr. 2787 (1973) .
L.
T. B. SANTULLI et al.,
N. Eng. J. Mad. 938-942 (1971) .
Fifth Inter. Cong. 317-340. Eds.
N. M. HUNTER and J. S. GLOVER, Biochem . J. _89, Science 158,
114-123
1570-1572 (1967) .
S. FREEDMAN and M. BONNAY, Experientia 27, 632-633 (1971) . L. B. GEFFEN and S. UDENFRIEND, Fed.
Proc . 32, No . 3 .
Pergamon Press
A RADIOIMMUNOASSAY OF HUMAN CIRCULATORY DOPAMINE-ß-HYDFWXYLASIE Richard P. Ebstein,
Dong H. Park, Lewis S. Freedman, Stuart M. Levitz,
Takeshi Ohuchi and Menek Goldstein New York University Medical Center, Dep artment of Psychiatry Neurochemistry Laboratories, New York, N.Y. 10016
(Received 1 August 1973 ; in final form 21 August 1973) summary A procedure for the radioimmunoassay of human circulatory dopamine-ß-hydroxylase (DßH) using antibodies directed toward human DpH is described. A significant correlation between serum DßH ac tivity and the amount of serum immunoreactive (MR) DßH was found in the analyzed population . The radioimmunoassay of serum DßH could be used in monitoring changes of sympathetic activity in various physiological and pathological states . Introduction The levels of serum dopamine-ß-hydroxylass
(EC 1.14.2 .1) activity vary
widely in the normal population but are maintained at a relatively constant level in each individual
(1,2) .
The DßH activity in the serum may not reflect the
amount of the enzyme released from synaptic vesicles following sympathetic stimulation since the enzyme might be inactivated to some extent prior to its removal from the circulation.
The radioimmunoassay can be applied to the estimation of
the total IR enzyme-protein
(active and inactive enzyme) and this procedure is
not dependent on the labile enzyme activity.
Recently a solid-radioimmunoassay
utilizing antibodies to sheep adrenal DßH and 1 was described (3) .
125
labelled sheep adrenal DßH
Since there is a considerable cross species loss in i mn,n o-
reactivity (4) we have modified the radioimmunoassay for determination of circulatory human DßH by. using 1
125
labelled human DßH to inhibit competitively the
binding of human serum DßH to antibody directed toward human enzyme .
In this
communication we will describe the procedure for the determination of human serum 1 This work was supported by PHs Grant MH02717 and NSF Grant GB27603.
769
770
Assay for Dopamine-ß-Hydroxylase
Vol . 13, No. 7
DßH by the radioimmunoassay and we will compare the total serum
Ijpft
protein lev-
els with the serum DßH activity levels in humans . Methods DßH was purified from a human pheochromocytoma tumor .
The tumor was ob-
tained immediately after surgery and the enzyme was purified from the 100,000 x g particulate fraction by the previously described procedure (5) .
As a final puri
fication step the enzyme preparation was submitted to a linear sucrose density gradient centrifugation (Park,
Ebstein and Goldstein,
unpublished data) .
The
electrophoresic analysis of the final preparation after sucrose density gradient centrifugation showed only a single band of DßH.
Human antibodies against puri-
fied human DßH were produced in rabbits and tested for specificity by imm noelectrophoresis .
Following immunoelectrophoresis human antiserum gives a single
precipitin line with DßH preparations obtained from human pheochromocytomas but does not give a precipitin line with DßH preparations obtained from bovine adrenal glands . al,
(6),
DßH was iodinated for use as a tracer by the method of Greenwood et
To a solution of 25 Wg purified DßH in 0,5 M phosphate buffer at pH 7,5
was added 2 me of sodium iodide-125
(sp. act,
T in 10 ~L1 of phosphate buffer saline (PBS) . sec.
17 Ci/mg)
and 0.1 mg of chloramine-
The reaction was stopped after 30
by the addition of 0.2 mg sodium metabisulfite in 200 ~L1 of PBS and 2 mg of
KI in 0.2 ml was added.
The solution was passed through a column (1 x 22 cm) of
Sephadex G-25 equilibrated with PBS,
and the eluted iodine labelled protein was
then submitted to a linear sucrose density gradient (5-208 sucrose) . tion containing 1
125
labelled DßH was used for the radioimmunoassay .
The fracThe serum
DßH levels were measured by the solid-state radioimmunoassay as described by Catt and Tregear (7) .
1
125
labelled DßH was incubated in antibody-coated tubes
for 48 hrs with either standard amounts of purified DßH or serum samples. rinsing with water,
After
the dry tubes were counted in a well counter (Nuclear Chicago,
1185 Automatic Gamma Counter ),
DßH activity in the serum was determined by the
previously described procedure (8),
Vol. 13, No. 7
Assay for Dopamine-ß-Hydroxylase Fig.
1.
9
7
a u 5
4
3
1 1 . 0
10
.
20 rig DOFI
.
30
40
standard radioiauunoassay curve for . human DPI Fig.
Z.
50r-
40
É
30
10
0
20
40 60 80 Enzymatic activity (units)
100
The relationship between serum DßH activity and serum üt DßH in humans. The correlation coefficient - 0 .80 P < .01 (N-60)
771
77 2
Vol. 13, No . 7
Assay for Dopamine -ß-Hydroxylase TABLE 1 The Relationship of Human Serum DßH Activity to Immunoreactant DßH Levels
No. of Subjécts *
IR DßH ***
DßH Activity** (range)
4g/ml
19
0-20
6.1
± 0.69
21
21-40
11.6
± 1.09
20
41-90
20 .49 ± 1.67
There was no significant difference between the analyzed normal children (age group S-16) and analyzed normal adults in the mean values of the serum IR DßH amounts and in the serum DßH activities . One unit is defined as the enzyme activity that foams 1 nmole of C14-N-methylated product per ml of serum per 20 min incubation time . ***
These results represent the means ± S.E .M.
Results Figure 1 shows the standard radioincwso assay curve for human DPI, tion of 1
125
labelled DßH was linear over the range 1-20 ng DPIL
Inhibi
The standard
error of the mean after four determinations on a single sample was 5% of the mean assay.
Several dilutions of human serum and standard preparations of purified
human DßH showed parallel decreases in inhibition.
The total circulatory IR an-
zyme-protein levels and the enzyme activity levels were analyzed in 35 normal individuals and in 25 individuals with various mental and neurological disorders of both sexes and various age groups .
Serum IR enzyme-protein levels vary among the
analyzed individuals but the range of values is narrower than that of the enzymatic activities .
There is a preponderance of sera with low IR enzyme-protein
levels in the low enzyme activity group and a preponderance of sera with high IR enzyme-protein levels in the high enzyme activity group.
The differences in the
mean values of serum IR enzyme-protein levels between different enzyme activity
Assay for Dopamine -ß- Hydroxylase
Vol . 13, No. 7
groups is statistically significant (Table 1) .
773
A plot of enzyme activity levels
versus IR enzyme-protein levels reveals that there is a significant correlation between enzyme activity levels and IR enzyme-protein levels (Fig .
2) .
When the
enzyme activity levels versus IR enzyme-protein levels were separately plotted for the normal population and for the population consisting of mental and neurological patients similar correlation coefficients were obtained . Discussion The present study describes a radio immun oassay for determination of human circulatory DßH.
125 This assay employs 1 labelled human DßH and antibodies di-
rected toward human DßH, while the previously described assay (3) employs 1 labelled sheep DßH and antibodies directed toward sheep DßH.
125
Thus, the lower
values obtained for human serum IR DßH by the previously described radioimmunoassay (3) as compared with the presently described assay might be due to a loss of cross-immunoreactivity between species.
It is of interest to note that with
the sheep radioimmunoassay system no correlation between human serum IR enzymeprotein levels and enzyme activity was found (9) .
In this study we have shown a
significant correlation between human serum IR enzyme-protein levels and enzyme activity levels.
These findings indicate that the radioimmunoassay employed in
this study reflects the DßH levels in human serum.
Furthermore,
preliminary
studies show that the radioimmunoassay is reliable in detecting acute changes in sympathetic activity . The simultaneous determination of circulatory DßH activity and of total enzyme-protein levels could be advantageous .
A correlation,
or lack of correlation
between these two assays will indicate whether the alterations in circulatory en zyme levels are due to changes in the rate of enzyme release or to changes in the rate of enzyme inactivation prior to its elimination from the circulation.
In a
separate communication we will report on the circulatory human IR DßH levels and on enzyme activity levels in various physiological and pathological states .
774
Assay for Dopamine -ß- Hydroxylase
Vol . 13, No. 7
References 1.
J. F. LINGLEY, R H, SAGERMAN, 277, 1227-1230 (1967) .
2.
R M. WEINSHILBOUM and J. AXELROD, N. Eng. J. Med. 285,
3.
R. A. RUSH and L. H. GEFFEN, Cir. Res. 31, 444-452 (1972) .
4.
T. OHUCHI, T. H. JOH, L. S. FREEDMAN and M. GOLDSTEIN, on Pharmacol. Abstr. 1022, p. 171 (1972) .
5.
M. GOLDSTEIN, in : Methods in Neurochemistry, Chapter 13, pp. N. Marks and R Rodnight. Plenum Press, New York (1972) .
6.
F. C. GREENWOOD, (1963) .
7.
K. J. GATT and G. W. TREGEAR,
8.
M. GOLDSTEIN,
9.
R. A. RUSH, T. NAGATSU, Abstr. 2787 (1973) .
L.
T. B. SANTULLI et al.,
N. Eng. J. Mad. 938-942 (1971) .
Fifth Inter. Cong. 317-340. Eds.
N. M. HUNTER and J. S. GLOVER, Biochem . J. _89, Science 158,
114-123
1570-1572 (1967) .
S. FREEDMAN and M. BONNAY, Experientia 27, 632-633 (1971) . L. B. GEFFEN and S. UDENFRIEND, Fed.
Proc . 32, No . 3 .