Journal of Neurochernrstry, 1977. Vol. 29, pp. 1141-1142. Pergamon Press. Printed in Great Britain.
SHORT COMMUNICATION
Dopamine-/?-hydroxylaseactivity in human cerebrospinal fluid and serum (Received 15 February 1977. Revised 21 M a y 1977. Accepted 26 M a y 1977)
DOPAMINE-a-hydroxytase (DBH) in serum is secreted from peripheral sympathetic nerve terminals together with noradrenaline and may have some correlation with the activity of peripheral noradrenergic neurones (AXELROD, 1973). The enzyme has been found in rabbit (DE POTTER,1976) and & CUBEDDU, 1976; OKADA er al., 1976) human (GOLDSTEIN cerebrospinal fluid, and may be derived from central noradrenergic neurones. We have devised a highly sensitive new fluorometric assay for DBH activity by high-performance liquid chromatography (FUIITA et a]., in preparation), and applied this method to the assay of DBH activity in human cerebrospinal fluid. The DBH activity in human cerebrospinal fluid was compared with that in serum. Cerebrospinal fluid was obtained from patients at Fujita-Gakuen University School of Medicine Hospital by lumbar puncture. The patients were undergoing surgery under lumbar anesthesia. No patient suffering from central or peripheral neurological disease was included, and the general physical and nutritional states of the patients were within the normal range. The first 5ml was removed for chemical and cytological examination, and the next 5ml was used for the assay of b B H activity. The samples of cerebrospinal fluid were all clear, and no red cells were detected. Blood samples were obtained by venepuncture and serum was separated. A hhreriation used: DBH, dopamine-8-hydroxylase.
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0.4
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0.3
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DBH incubation mixture (total volume 1.0 ml) contained (in final concentrations): 400 p1 of cerebrospinal fluid or 50 pl of serum plus 350 p1 of water as enzyme, 0.2 M-acetate buffer (pH 5.0), 1 mM-pargyline HCI, 30 mM-N-ethylmaleimide, 20 ~M-CUSO,,10 mM-sodium fumarate, 10 mM-ascorbic acid, 500 pg (25,000 units) of catalase, and 20 mwtyramine HCI. Fusaric acid (0.1 mM) (HIDAKAet al., 1969; et al., 1970),a specific DBH inhibitor, was added NACATSU to the blank before incubation, and to the experimental tube after the incubation. After incubation, 200p1 of ~ M - T C Awas added to the reaction mixture which was centrifuged at 2500rev./min for 10min to remove protein and the supernatant was transferred to a small column (0.9 x 10.5cm) of Dowex 50W-X8 (H', 2 W 4 0 0 mesh, 0.98 g dry weight). The column was washed four times with 5 ml of 0.1 M-sodium phosphate buffer, pH 8.0, containing 0.05~-NaCIand four times with 5ml of water, and the adsorbed amines were eluted with 3.0 ml of 3.5% NH,OH into a test tube. The eluate was evaporated at 60°C and the residue was dissolved in 200p1 of the solvent and an aliquot of the solution, 10 pi, was subjected to a high-performance liquid chromatography (packing: p Bondapak C18, column: 1/4in x 12in, solvent: 5.25g of citric acid, 4.10 g of sodium acetate, 1.05 ml of acetic acid, 2.9 g of sodium hydroxide, 23.2g of sodium chloride per litre of deionized, distilled water purified through Milli Q'" system, pH 5.10 f 0.02, flow rate: 1 ml/min). The octopamine peak (retention time: 7 min) was analysed fluorometrically
E
\
3
E
LL
rn
0.1
0
.. .. . 20
40
60 SERUM
80 100 DBH ( r n U / r n l )
120
140
FIG. 1. Correlation between dopamine-8-hydroxylase (DBH) activity in serum and that in cerebrospinal fluid (CSF). The activity is expressed as mU (nmolmin-') ml-'. I141
1142
Short communication
& HARE,1975). Details of using o-phthalaldehyde (HENSON spinal fluid was increased by stimulating the peripheral this new assay method for DBH activity will be published (sciatic) nerves, and he suggested the possibility of release elsewhere. For measuring the DBH activity in cerebro- of DBH from central noradrenergic neurones. In the spinal fluid where the enzyme activity is very low, it was present human study, however, the correlation between found to be essential t o use fusaric acid for the blank t o cerebrospinal fluid and serum is too low to allow any firm obtain a low and reproducible blank value. When TCA conclusions to be drawn. was added before incubation (zero-time blank), the blank value increased slowly with time. Institute for Comprehensive K. FUJITA Since DBH activity in serum and cerebrospinal fluid Medical Science, K. MARUTA varied widely, a computer generation of ‘the clinical refer- School of Medicine, R. TERADAIRA (1963) erce values’, based on the program by HOFFMANN Fajita-Gakuen UniOersity. H. BEPPU was used for the statistical analysis. Toyoake, Aichi 470-1 1, Japan K . SHINPO DBH activity (mean s.D.) in cerebrospinal fluid Y. MAENO (1963) without the statistical computation by HOFFMANN T. IT0 was 0.1747 5 0.1654pnol/min per litre (n = 120, range 0.0086-0.9470) and after the computation was 0.1311 k Laboratory of Cell Physiology, T. NAGATSU 0.0763 pmol/min per litre (n = 108, range 0.0086-0.3788). Department of Life Chemistry, T. KATO DBH activity in cerebrospinal fluid and serum was Graduate Schuol at Nagatsuda, measured simultaneously in 54 patients. After the Hoff- Tokyo Institute of Technology, mann computation, 8 cases with abnormally high or low Tokyo 152, Japan DBH activity in cerebrospinal fluid and/or serum were rejected, and the remaining 46 cases were used t o calculate the correlation between the DBH activity in cerebrospinal REFERENCES fluid and that in serum. The DBH activity in cerebrospinal fluid and in serum was 0.1272 k 0.0707 and AXELRODJ. (1973) Pharmac. Rev. 25, 233-243. s.D.), respect41.43 31.02/~mol/minper litre (mean DE POTTERW. P. (1976) J. Physiol., Lond. 258, 26p27p. ively. As shown in Fig. 1, a correlation coefficient between GOLDSTEIN D. J. & CUBEDDUL. X. (1976) J . Neurochem. the DBH activity in cerebrospinal fluid and that in serum 26, 193-195. was 0.2926 (P < 0.05). HENSONJ. R. & HAREP. E. (1975) Proc. natn. Acad. Sci., Serum DBH may be mostly derived from the nerve terU.S.A. 72, 619-622. minals of the peripheral sympathetic nerves (AXELROD, HIDAKAH., NAGATSU T., TAKEYA K., TAKEUCHI T., SUDA 1973). The origin of DBH in cerebrospinal fluid is unH., KOJIRIK.. MATSUZAKI M. & UMEZAWA H. (1969) known. It may also be derived from the nerve terminals J . Antibiotics 22, 228-230. of the central noradrenergic neurones. However, cerebro- HOFFMANN R. J . (1963) J . Am. med. Assoc. 185, 864-873. spinal fluid levels are less than 1% of those in serum. NAGATSU T., HIDAKA H., KUZUYAH.. UMEZAWA H., TAKTherefore, this low level could possibly have been derived EUCHl T.& SUDA H. (1970) Biorhem. Pharmac. 19, 35-44. in part from blood by contamination of the lumbar punc- OKADAT., OHTAT., SHINODA T., KATOT., IKUTA K. & (1976) found that DBH in rabbit cerebroture. DE POTTER NAGATSU T. (1976) Neuropsychobiology 2, 139-144.
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SHORT COMMUNICATION
Dopamine-/?-hydroxylaseactivity in human cerebrospinal fluid and serum (Received 15 February 1977. Revised 21 M a y 1977. Accepted 26 M a y 1977)
DOPAMINE-a-hydroxytase (DBH) in serum is secreted from peripheral sympathetic nerve terminals together with noradrenaline and may have some correlation with the activity of peripheral noradrenergic neurones (AXELROD, 1973). The enzyme has been found in rabbit (DE POTTER,1976) and & CUBEDDU, 1976; OKADA er al., 1976) human (GOLDSTEIN cerebrospinal fluid, and may be derived from central noradrenergic neurones. We have devised a highly sensitive new fluorometric assay for DBH activity by high-performance liquid chromatography (FUIITA et a]., in preparation), and applied this method to the assay of DBH activity in human cerebrospinal fluid. The DBH activity in human cerebrospinal fluid was compared with that in serum. Cerebrospinal fluid was obtained from patients at Fujita-Gakuen University School of Medicine Hospital by lumbar puncture. The patients were undergoing surgery under lumbar anesthesia. No patient suffering from central or peripheral neurological disease was included, and the general physical and nutritional states of the patients were within the normal range. The first 5ml was removed for chemical and cytological examination, and the next 5ml was used for the assay of b B H activity. The samples of cerebrospinal fluid were all clear, and no red cells were detected. Blood samples were obtained by venepuncture and serum was separated. A hhreriation used: DBH, dopamine-8-hydroxylase.
--
0.4
-
0.3
-
DBH incubation mixture (total volume 1.0 ml) contained (in final concentrations): 400 p1 of cerebrospinal fluid or 50 pl of serum plus 350 p1 of water as enzyme, 0.2 M-acetate buffer (pH 5.0), 1 mM-pargyline HCI, 30 mM-N-ethylmaleimide, 20 ~M-CUSO,,10 mM-sodium fumarate, 10 mM-ascorbic acid, 500 pg (25,000 units) of catalase, and 20 mwtyramine HCI. Fusaric acid (0.1 mM) (HIDAKAet al., 1969; et al., 1970),a specific DBH inhibitor, was added NACATSU to the blank before incubation, and to the experimental tube after the incubation. After incubation, 200p1 of ~ M - T C Awas added to the reaction mixture which was centrifuged at 2500rev./min for 10min to remove protein and the supernatant was transferred to a small column (0.9 x 10.5cm) of Dowex 50W-X8 (H', 2 W 4 0 0 mesh, 0.98 g dry weight). The column was washed four times with 5 ml of 0.1 M-sodium phosphate buffer, pH 8.0, containing 0.05~-NaCIand four times with 5ml of water, and the adsorbed amines were eluted with 3.0 ml of 3.5% NH,OH into a test tube. The eluate was evaporated at 60°C and the residue was dissolved in 200p1 of the solvent and an aliquot of the solution, 10 pi, was subjected to a high-performance liquid chromatography (packing: p Bondapak C18, column: 1/4in x 12in, solvent: 5.25g of citric acid, 4.10 g of sodium acetate, 1.05 ml of acetic acid, 2.9 g of sodium hydroxide, 23.2g of sodium chloride per litre of deionized, distilled water purified through Milli Q'" system, pH 5.10 f 0.02, flow rate: 1 ml/min). The octopamine peak (retention time: 7 min) was analysed fluorometrically
E
\
3
E
LL
rn
0.1
0
.. .. . 20
40
60 SERUM
80 100 DBH ( r n U / r n l )
120
140
FIG. 1. Correlation between dopamine-8-hydroxylase (DBH) activity in serum and that in cerebrospinal fluid (CSF). The activity is expressed as mU (nmolmin-') ml-'. I141
1142
Short communication
& HARE,1975). Details of using o-phthalaldehyde (HENSON spinal fluid was increased by stimulating the peripheral this new assay method for DBH activity will be published (sciatic) nerves, and he suggested the possibility of release elsewhere. For measuring the DBH activity in cerebro- of DBH from central noradrenergic neurones. In the spinal fluid where the enzyme activity is very low, it was present human study, however, the correlation between found to be essential t o use fusaric acid for the blank t o cerebrospinal fluid and serum is too low to allow any firm obtain a low and reproducible blank value. When TCA conclusions to be drawn. was added before incubation (zero-time blank), the blank value increased slowly with time. Institute for Comprehensive K. FUJITA Since DBH activity in serum and cerebrospinal fluid Medical Science, K. MARUTA varied widely, a computer generation of ‘the clinical refer- School of Medicine, R. TERADAIRA (1963) erce values’, based on the program by HOFFMANN Fajita-Gakuen UniOersity. H. BEPPU was used for the statistical analysis. Toyoake, Aichi 470-1 1, Japan K . SHINPO DBH activity (mean s.D.) in cerebrospinal fluid Y. MAENO (1963) without the statistical computation by HOFFMANN T. IT0 was 0.1747 5 0.1654pnol/min per litre (n = 120, range 0.0086-0.9470) and after the computation was 0.1311 k Laboratory of Cell Physiology, T. NAGATSU 0.0763 pmol/min per litre (n = 108, range 0.0086-0.3788). Department of Life Chemistry, T. KATO DBH activity in cerebrospinal fluid and serum was Graduate Schuol at Nagatsuda, measured simultaneously in 54 patients. After the Hoff- Tokyo Institute of Technology, mann computation, 8 cases with abnormally high or low Tokyo 152, Japan DBH activity in cerebrospinal fluid and/or serum were rejected, and the remaining 46 cases were used t o calculate the correlation between the DBH activity in cerebrospinal REFERENCES fluid and that in serum. The DBH activity in cerebrospinal fluid and in serum was 0.1272 k 0.0707 and AXELRODJ. (1973) Pharmac. Rev. 25, 233-243. s.D.), respect41.43 31.02/~mol/minper litre (mean DE POTTERW. P. (1976) J. Physiol., Lond. 258, 26p27p. ively. As shown in Fig. 1, a correlation coefficient between GOLDSTEIN D. J. & CUBEDDUL. X. (1976) J . Neurochem. the DBH activity in cerebrospinal fluid and that in serum 26, 193-195. was 0.2926 (P < 0.05). HENSONJ. R. & HAREP. E. (1975) Proc. natn. Acad. Sci., Serum DBH may be mostly derived from the nerve terU.S.A. 72, 619-622. minals of the peripheral sympathetic nerves (AXELROD, HIDAKAH., NAGATSU T., TAKEYA K., TAKEUCHI T., SUDA 1973). The origin of DBH in cerebrospinal fluid is unH., KOJIRIK.. MATSUZAKI M. & UMEZAWA H. (1969) known. It may also be derived from the nerve terminals J . Antibiotics 22, 228-230. of the central noradrenergic neurones. However, cerebro- HOFFMANN R. J . (1963) J . Am. med. Assoc. 185, 864-873. spinal fluid levels are less than 1% of those in serum. NAGATSU T., HIDAKA H., KUZUYAH.. UMEZAWA H., TAKTherefore, this low level could possibly have been derived EUCHl T.& SUDA H. (1970) Biorhem. Pharmac. 19, 35-44. in part from blood by contamination of the lumbar punc- OKADAT., OHTAT., SHINODA T., KATOT., IKUTA K. & (1976) found that DBH in rabbit cerebroture. DE POTTER NAGATSU T. (1976) Neuropsychobiology 2, 139-144.
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