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Protection of endogenous enkephalin degradation from peptidases in human serum by actinonin
Neuropephdes (1989) 13,201-206 @ Longman Group UK Ltd 1989
0143-4179/89/0013-0201/$10.00
Protection of Endogenous Enkephalin Degradation from Peptidases in Human Serum by Actinonin S. NAKAJIMA”,
K. KAYA” and T. HAZATOt
*Department of Anesthesiology, School of Medicine, Juntendo University, 3-I-3, Hongo, Bunkyo-ku, Tokyo, 113, Japan. t Department of Medical Chemistry, The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 173, Japan (reprint requests to S. N.).
Abstract-This paper describes a new method for detecting the endogenous enkephalin level in human serum without interference from serum peptidases and contaminating proteins. The method consists of a combination of addition of a peptidase inhibitor, actinonin to serum and dialysis. The activity of enkephalin degrading enzymes was completely abolished by actinonin. Furthermore, contaminating proteins which interfered with RIA assay of enkephalins were removed by the dialysis process. This combination process enabled precise detection of the concentration of enkephalin-like substances in human serum. The mean normal serum level of leucine-enkephalin assayed by this method in 12 healthy volunteers was 93.4pg/ml.
Introduction After the discovery of methionine-enkephalin (Met-enkephalin) and leucine-enkephalin (Leuenkephalin) in 1975 (l), many opioid peptides were isolated from various organs. Recent advances in immunoassay have shown that enkephalin-like peptides are widely distributed in an extensive range of organs and tissues such as the pituitary body (2,3), small intestine (4) and adrenal gland (5). They may act as neurotransmitters or neuromodulators in the central and peripheral nervous system (6,18) and mediators of Date received 8 December 1988 Date accepted 12 December 1988
the central nervous system’s effects on the immune system (7). Met- and Leu-enkephalins have been determined in both human serum and plasma (8,9), and different values have been reported for their concentrations (10, 11, 12,13,14). One reason for the difference may be the presence of enzymes in serum or plasma which degrade enkephalins to immunoinactive forms. Those enzymes are thought to be aminopeptidases, dipeptidyl aminopeptidase and dipeptidyl carboxypeptidase. Furthermore, we reported that opioid peptides such as Leu-enkephalin in human serum were bound to a protein having a molecular weight of 400000 (15). The protein seemed to store opioid peptides and modulate their metabolism. This 201
202 suggests that opioid peptides do not exist in a free state in the serum. In this report, we found that addition of actinonin to serum effectively prevented enkephalin degradation while Leu-enkephalin was being separated from contaminating proteins in serum by dialysis. This permitted its detection at a lower level than previous methods. By use of this method, we report Leu-enkephalin levels in healthy volunteers are higher than those reported to date.
NEUROPEPTIDES
Sample or standard Leu-enkephalin
lOO/lI
[1261] Leusnkephalin
100~1
Anti-Leu-enkephalin
(10 000 cpm) serum
100 /.I1
Incubate at 4”C, 24 hr + 100 1.111.0% Bovine y-globulin + 500 ~1 30% Polyethylene glycol6000 Stir Stron&
10 sec.
Centrifuge at 3 000 rpm for 10 min.
Precipitate
I
Materials and Methods Materials
Leu-enkephalin and actinonin (16) were obtained from Peptide Institute Inc., Osaka, Japan. [12’1] Leu-enkephalin was purchased from New England Anti-Leu-enkephalin Nuclear, Mass., U.S.A. serum was prepared according to the method described by Kuraishi et al. (17). An ODS-Q3 column, bovine y-globulin and polyethylene glycol 6000 were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan.
Serum
Blood was drawn from the cubital vein of 10 healthy men and 2 healthy women in their thirties to fifties. After actinonin was added to the blood to a concentration of lOO~g/ml, the mixture was allowed to stand for 30 min at room temperature and then centrifuged at 3000 rpm for 20 min at 4°C to obtain the serum.
Dialysis and separation of Leu-enkephalin
One ml of serum (from blood to which actinonin was added to a concentration of lOOpg/ml) was dialyzed against 100ml of 25mM Tris-HCl buffer (pH 7.0) for 24hr. The dialysate was applied to a silica gel ODS-Q3 column, and the Leu-enkephalin fraction was eluted with 80% methanol. The Leu-enkephalin fraction was then evaporated to dryness and assayed by the RIA method. As controls, serum which contained no actinonin and was not dialyzed were also employed.
Count the redioactivitv
Figure
Procedure of the radioimmunoassay
enkephalin
method of Leu-
RZA (Radioimmunoassay) The figure shows the radioimmunoassay method of Leu-enkephalin. One hundred ~1 of test sample, 100~1 of [1251] Leu-enkephalin (10OOOcpm) and 100~1 of antiLeu-enkephalin serum were mixed and incubated for 24hr at 4°C. After the incubation, 100~1 of 1.0% bovine y-globulin and then 500~1 of 30% polyethylene glycol 6000 were added to the mixture and mixed well for 10 sec. The reaction mixture was centrifuged at 3000 rpm for 10 min and the radioactivity in the precipitate was measured by counting in a liquid scintillation counter (Model LSC-700, Aloka Co., Ltd., Tokyo, Japan). Standard curves were plotted using standard Leu-enkephalin treated by the same method as described above. The Leu-enkephalin concentration in each sample was determined from the standard curve. Identification of Leu-enkephalin by high performance liquid chromatography (HPLC)
HPLC analysis was carried out using 1% acetic acid/acetonitrile (75:25, v/v) as a developing solvent. The column was used p-Bondapack Cl8 (Waters Associates, Inc., Milford, Mass., U.S.A., 7.8mm x 30cm) and the detector was Millipore Waters Model 440. Absorbance was measured at 280nm and the flow rate was 2mYmin.
203
ENDOGENOLJSENKEPHALIN-DEGRADATION
Effect of dialysis on recovery rate of Leu-enkephalin exogenously added to serum Table 1 shows that about 80% of Leu-enkephalin was recovered from serum to which actinonin had not been added and which had not been dialyzed when Leu-enkephalin was exogenously added to serum at concentrations of 11.1 and 99.7pglml. The endogenous Leu-enkephalin level in untreated serum was 4Spg/ml. These data indicate that the recovery rate is rather high when Leu-enkephalin is added to serum and dialysis is not performed. Recovery of Leu-Enkephahn Exogenously added to Serum without Dialysis and without Actinonin Table 1
Added Leu-enkephalin
Concentration of Endogenous Leu-Enkephalin in Serum with or without Dialysis Table 2
Results
Measured
Recovered
Recovery
(pglml)
(pglml)
(pglml)
00)
0 11.1 99.7
4.5 13.5 86.7
9.0 82.2
81.1 82.4
The detected endogenous serum level of Leuenkephalin was increased from 4Spg/ml to 34.3pg/ml when the same serum was dialyzed against 25mM Tris/HCl buffer (pH 7.0) without addition of actinonin, as shown in Table 2. This experiment was repeated 7 times. Effect of actinonin addition and dialysis of serum Addition of actinonin to blood and dialysis of the
Leu-enkephalin
Experiment No.
(pglml)
without dialysis
with dialysis
1 2 3 4 5 6 7
4.7 3.6 5.4 3.7 4.0 3.4 6.7
40.9 36.7 30.1 28.8 30.9 37.9 36.1
Mean + SD
4.5 * 1.1
34.3 + 4.2
No actinonin was added to the serum.
serum increased the recovery of Leu-enkephalin as shown in Table 3. Leu-enkephalin was added to actinonin containing serum at a concentration of 50.4pg/ml followed by incubation at 4°C or 37°C for 3 hr. After that, the serum was dialyzed, and Leu-enkephalin was assayed by the same method as described above. The Leu-enkephalin recovery from the serum was 25.6% and 21.1% without addition of actinonin when it was incubated for 3 hr at 4°C or 37°C before dialysis respectively. On the other hand, the recovery rates were 90.3% and 89.9% when actinonin was added to the serum. There was no difference in the recovery rates with incubation at 4°C and 37°C. These data suggest that actinonin addition effectively prevented degradation of Leu-enkephalin in the serum during dialysis and increased the recovery rate from 25.6% to 90.3%.
Effect of Actinonin on Exogenously added Leu-Enkephalin Recovery at 4°C or 37°C Incubation in Serum before Dialysis
Table 3
Actinonin (Wglm!, 0
Incubation (3 hrs) 4°C 37°C 4°C
100 37°C
Added
Leu-enkephalin Measured
0
59.8
50.4 0 50.4 0
72.7 59.2 69.9 75.6 121.1 63.7 109.0
50.4 0 50.4
(pglml)
Recovery Recovered
(%) _
12.9
25.6
10.7
21.2
45.5
90.3
45.3
89.9
204
NEUROPEPTIDES
Serum Level of Leu-Enkephalin in Healthy Volunteers Table 4
Leu-enkephalin Actinonin
Dialysis
0kg/m1 lOOpg/ml
(+I (+I
0 t&ml
C-1
Leu-enkephalin
(P&4 21.1 93.4 1.0
level in serum of healthy volunteers
Table 4 shows the levels of Leu-enkephalin in the serum of the 12 healthy volunteers. The serum level of Leu-enkephalin was 93.4pgl ml when actinonin was added to blood to a concentration of lOO~g/ml. The serum Leu-enkephalin concentration without addition of actinonin was 2l.lpg/ml. Leu-enkephalin was 7.0pg/ml when it was assayed without actinonin and without dialysis. HPLC
analysis
The eluted sample was fractionated at 2ml/min and each of the fractions was assayed for Leuenkephalin concentration by the RIA method described above. The retention time of Leu-enkephalin from the samples was 17 min. Standard Leu-enkephalin had a retention time of 17 min in the same condition.
Discussion This paper describes a new method for detection of Leu-enkephalin in human serum by combination of the processes of dialysis and addition of an appropriate peptidase inhibitor, actinonin. Actinonin shows strong competitive inhibition with substrates for enkephalin-aminopeptidase, dipeptidyl aminopeptidase and enkephalinase A (20). Since human blood contains various peptidases, it was hypothesized that actinonin might protect Leu-enkephalin from degradation by these peptidases. On the other hand, dialysis is an efficient process for detecting Leu-enkephalin, as shown in Tables 1 and 2. The concentration in serum was increased 7.6 times by this process, as shown in Table 2.
The Leu-enkephalin plasma level in healthy persons was reported to be 54 f 10 (average + standard deviation) pg/ml by Ryder et al. (10) and below 3pg/ml by Yoshimasa et al. (12). However, we found the serum Leu-enkephalin level in 12 healthy volunteers using the present method to be 93.4 pg/ml, which is 1.73 times higher than Ryder et al’s value for 9 fasting healthy subjects (10). Both Ryder et al. and Yoshimasa et al. acidified the plasma to protect Leu-enkephalin from proteolytic enzymes, however, they assayed it by RIA methods without dialysis of the plasma. Their low value of the Leu-enkephalin level suggests that acidity failed to inactivate proteolytic enzymes completely or the enkephalin binding protein did not release Leu-enkephalin. Aloyo et al. (19) said that they could not completely inhibit Met-enkephalin breakdown with bestatin, an aminopeptidase inhibitor. They (19) suggested that bestatin did not inhibit aminopeptidase activity completely or that peptidases other than aminopeptidase were active in the blood and plasma. Thus, we felt that another better peptidase inhibitor might improve the detection of endogenous enkephalin-like substances. In our experiment, actinonin was used at a concentration of lOOl~,g/ml, which is 256 times more than its ICso for enkephalin-aminopeptidase, 91 times more than its ICSOfor dipeptidyl aminopeptidase and 18 times more than its IGO for enkephalinase A (20). The serum level of Leu-enkephalin was 93.4pg/ ml when assayed by our method, as shown in Table 4. On the contrary, the same serum showed 21.1 pg/ml when it was assayed after dialysis but without addition of actinonin. These data indicate that the combined process of addition of actinonin and dialysis is useful for detection of the Leu-enkephalin level in human serum. Our findings suggest that the difference between 93.4pg/ml with actinonin and 21.1 pg/ml without it is due to degradation by aminopeptidases in the serum during dialysis. Table 3 shows that pre-incubation temperatures of 4°C and 37°C gave equivalent results; therefore, it appears that exogenously-added Leu-enkephalin is not degraded in the serum during the incubation. Table 1 shows that exogenously-added Leu-enkephalin was recovered at rather high rates
205
ENDOGENOIJS ENKEPHALIN-DEGRADATION
from serum without either dialysis or actinonin, although the endogenous Leu-enkephalin concentration was as low as 4Spgfml. On the other hand, Table 3 shows that the recovery rate of exogenously-added Leu-enkephalin was reduced to about 25% by the process of dialysis when no actinonin was added to the serum. Addition of actinonin increased the recovery to about 90%. In the present method, actinonin seems to have protected Leu-enkephalin from aminopeptidases in the serum during dialysis for the long period of 24hr. We confirmed that the HPLC column retention time of Leu-enkephalin from the serum was identical with that of standard Leu-enkephalin. Since actinonin exerts strong competitive inhibitory activity against enkephalin-aminopepand dipeptidyl aminopeptidase tidase , enkephalinase A, it is hypothesized that addition of actinonin in serum will also be useful for detection of serum level of enkephalin-like substance other than Leu-enkephalin in man and animals. Use of the method presented here to accurately determine the serum level of opioid peptides in normal subjects and patients with various diseases will help elucidate the mechanisms and roles of opioid peptides in man and animals in the future.
5.
6.
7.
8.
9
10.
11
12
13
Acknowledgements The authors wish to express a special gratitude to Biomedical Laboratories for their assistance in the Leu-enkephalin assay.
14.
References 15. Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. andMorris, H. R. (1975). Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258: 577-579. Li, C. H. and Chung, D. (1976). Isolation and structure of an untriakontapeptide with opiate activity from camel pituitary glands. Proceedings of the National Academy of Sciences of the United States of America 73: 1145-1148. Goldstein, A. (1976). Opioid peptides (endorphins) in pituitary and brain. Science 193: 1081-1086. Schultzberg, M., Dreyfus, C. F., Gershon, M. D., Hbkfelt, T.. Elde, R. P., Nilsson, G., Said, S. and Goldstein, M. (1978) _Vip- , enkephalin- , substance, P- , and somatostatine-like immunoreactivity in neurons intrinsic to the
16.
17.
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intestine: immunohistochemical evidence from organotypic tissue cultures. Brain Research 155: 239-248. Schultzberg, M., Hokfelt, T., Lundberg, J. M., Terenius, L., Elfvin, L.-G. and Elde, R. (1978), Enkephahn-like immunoreactivity in nerve terminals in sympathetic ganglia and adrenal medulla and in adrenal medullary gland cells. Acta Physiologica Scandinavia 103: 475-477. Frederickson, R. C. A. (1977). Enkephalin pentapeptides - a review of current evidence for a physiological role in vertebrate neurotransmission. Life Sciences 21: 23-42. Morley, J. E., Kay, N. E., Solomon, G. F. and Plotnikoff. N. P. (1987). Neuropeptides: conductors of the immune orchestra. Life Sciences 41: 527-544. Hambrook, J. M., Morgan, B. A., Rance, M. J. and Smith, C. F. C. (1976). Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates. Nature 262: 782-783. Clement-Jones, V., Lowry, P. J., Rees, L. H. and Besser, G. M. (1980). Met-enkephalin circulates in human plasma. Nature 283: 295-297. Ryder, S. W. and Eng, J. (1981). Radioimmunoassay of leucine-enkephalin-like substance in human and canine plasma. Journal of Clinical Endocrinology and Metabolism 52: 367-369. Clement-Jones, V., Lowry, P. J., Rees. L. H. and Besser, G. M. (1980). Development of a specific extracted radioimmunoassay for methionine enkephalin in human plasma and cerebrospinal fluid. Journal of Endocrinology 86: 231-243. Yoshimasa, T., Nakao, K., Li, S., Ikeda, Y.. Suda, M., Sakamoto, M. and Imura, H. (1983). Plasma methionineenkephalin and leucine-enkephalin in normal subjects and patients with pheochromocytoma. Journal of Clinical Endocrinology and Metabolism 57: 706-712. Govoni, S., Bosio. A., Di Monda, E., Fazzari, G., Spano, P. F. and Trabucchi. M. (1983). Immunoreactive metenkepahlin plasma concentrations in chronic alcoholics and in children born from alcoholic mothers. Life Sciences 33: 1581-1586. Howlett, T. A., Tomlin, S., Ngahfoong, L.. Rees, L. H.. Bullen, B. A., Skrinar, G. S. and McArthur, J. W. (1984). Release of 8 endorphin and met-enkephalin during exercise in normal women: response to training. British Medical Journal 288: 1950-1952. Hazato. T., Shimamura, M., Hirose, A. and Katayama. T. (1985). Opioid peptide-binding protein in human serum: its relationship to the enkephalin-degrading enzymes. Japanese Journal of Pharmacology 39: 142P. Umezawa, H., Aoyagi, T.. Tanaka, T., Suda. H.. Okuyama, A., Naganawa, H., Hamada, M. and Takeuchi, T. (1985). Production of actinonin, an inhibitor of aminopeptidase M, by Actinomycetes. Journal of Antibiotics 38: 1629-1630. Kuraishi, Y., Sugimoto, M.. Hirota, N. and Takagi, H. (1982). Lack of effect of morphine and noxious stimuli on the met-enkephalin content in the spinal dorsal horn and nucleus reticularis gigantocellularis of the rat. Life Sciences 30: 2071-2077. Viveros, 0. H.. Diliberto, E. J. Jr.. Hazum. E. and Chang,
206 K.-J. (1979). Opiate-like materials in the adrenal medulla: evidence for storage and secretion with catecholamines. Molecular Pharmacology 16: 1101-1108. 19. Aloyo, V. J., Mousa, S. A. and Van Loon, G. R. (1986). Stabilization of methionine-enkephalin in human and rat blood. Life Sciences 39: 21-28.
NEUROPEPTIDES
20. Hachisu, M., Hiranuma, T., Murata, S., Aoyagi, T. and Umezawa, H., (1987). Analgesic effect of actinonin, a new potent inhibitor of multiple enkephalin degrading enzymes. Life Sciences 41: 235-240.
0143-4179/89/0013-0201/$10.00
Protection of Endogenous Enkephalin Degradation from Peptidases in Human Serum by Actinonin S. NAKAJIMA”,
K. KAYA” and T. HAZATOt
*Department of Anesthesiology, School of Medicine, Juntendo University, 3-I-3, Hongo, Bunkyo-ku, Tokyo, 113, Japan. t Department of Medical Chemistry, The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 173, Japan (reprint requests to S. N.).
Abstract-This paper describes a new method for detecting the endogenous enkephalin level in human serum without interference from serum peptidases and contaminating proteins. The method consists of a combination of addition of a peptidase inhibitor, actinonin to serum and dialysis. The activity of enkephalin degrading enzymes was completely abolished by actinonin. Furthermore, contaminating proteins which interfered with RIA assay of enkephalins were removed by the dialysis process. This combination process enabled precise detection of the concentration of enkephalin-like substances in human serum. The mean normal serum level of leucine-enkephalin assayed by this method in 12 healthy volunteers was 93.4pg/ml.
Introduction After the discovery of methionine-enkephalin (Met-enkephalin) and leucine-enkephalin (Leuenkephalin) in 1975 (l), many opioid peptides were isolated from various organs. Recent advances in immunoassay have shown that enkephalin-like peptides are widely distributed in an extensive range of organs and tissues such as the pituitary body (2,3), small intestine (4) and adrenal gland (5). They may act as neurotransmitters or neuromodulators in the central and peripheral nervous system (6,18) and mediators of Date received 8 December 1988 Date accepted 12 December 1988
the central nervous system’s effects on the immune system (7). Met- and Leu-enkephalins have been determined in both human serum and plasma (8,9), and different values have been reported for their concentrations (10, 11, 12,13,14). One reason for the difference may be the presence of enzymes in serum or plasma which degrade enkephalins to immunoinactive forms. Those enzymes are thought to be aminopeptidases, dipeptidyl aminopeptidase and dipeptidyl carboxypeptidase. Furthermore, we reported that opioid peptides such as Leu-enkephalin in human serum were bound to a protein having a molecular weight of 400000 (15). The protein seemed to store opioid peptides and modulate their metabolism. This 201
202 suggests that opioid peptides do not exist in a free state in the serum. In this report, we found that addition of actinonin to serum effectively prevented enkephalin degradation while Leu-enkephalin was being separated from contaminating proteins in serum by dialysis. This permitted its detection at a lower level than previous methods. By use of this method, we report Leu-enkephalin levels in healthy volunteers are higher than those reported to date.
NEUROPEPTIDES
Sample or standard Leu-enkephalin
lOO/lI
[1261] Leusnkephalin
100~1
Anti-Leu-enkephalin
(10 000 cpm) serum
100 /.I1
Incubate at 4”C, 24 hr + 100 1.111.0% Bovine y-globulin + 500 ~1 30% Polyethylene glycol6000 Stir Stron&
10 sec.
Centrifuge at 3 000 rpm for 10 min.
Precipitate
I
Materials and Methods Materials
Leu-enkephalin and actinonin (16) were obtained from Peptide Institute Inc., Osaka, Japan. [12’1] Leu-enkephalin was purchased from New England Anti-Leu-enkephalin Nuclear, Mass., U.S.A. serum was prepared according to the method described by Kuraishi et al. (17). An ODS-Q3 column, bovine y-globulin and polyethylene glycol 6000 were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan.
Serum
Blood was drawn from the cubital vein of 10 healthy men and 2 healthy women in their thirties to fifties. After actinonin was added to the blood to a concentration of lOO~g/ml, the mixture was allowed to stand for 30 min at room temperature and then centrifuged at 3000 rpm for 20 min at 4°C to obtain the serum.
Dialysis and separation of Leu-enkephalin
One ml of serum (from blood to which actinonin was added to a concentration of lOOpg/ml) was dialyzed against 100ml of 25mM Tris-HCl buffer (pH 7.0) for 24hr. The dialysate was applied to a silica gel ODS-Q3 column, and the Leu-enkephalin fraction was eluted with 80% methanol. The Leu-enkephalin fraction was then evaporated to dryness and assayed by the RIA method. As controls, serum which contained no actinonin and was not dialyzed were also employed.
Count the redioactivitv
Figure
Procedure of the radioimmunoassay
enkephalin
method of Leu-
RZA (Radioimmunoassay) The figure shows the radioimmunoassay method of Leu-enkephalin. One hundred ~1 of test sample, 100~1 of [1251] Leu-enkephalin (10OOOcpm) and 100~1 of antiLeu-enkephalin serum were mixed and incubated for 24hr at 4°C. After the incubation, 100~1 of 1.0% bovine y-globulin and then 500~1 of 30% polyethylene glycol 6000 were added to the mixture and mixed well for 10 sec. The reaction mixture was centrifuged at 3000 rpm for 10 min and the radioactivity in the precipitate was measured by counting in a liquid scintillation counter (Model LSC-700, Aloka Co., Ltd., Tokyo, Japan). Standard curves were plotted using standard Leu-enkephalin treated by the same method as described above. The Leu-enkephalin concentration in each sample was determined from the standard curve. Identification of Leu-enkephalin by high performance liquid chromatography (HPLC)
HPLC analysis was carried out using 1% acetic acid/acetonitrile (75:25, v/v) as a developing solvent. The column was used p-Bondapack Cl8 (Waters Associates, Inc., Milford, Mass., U.S.A., 7.8mm x 30cm) and the detector was Millipore Waters Model 440. Absorbance was measured at 280nm and the flow rate was 2mYmin.
203
ENDOGENOLJSENKEPHALIN-DEGRADATION
Effect of dialysis on recovery rate of Leu-enkephalin exogenously added to serum Table 1 shows that about 80% of Leu-enkephalin was recovered from serum to which actinonin had not been added and which had not been dialyzed when Leu-enkephalin was exogenously added to serum at concentrations of 11.1 and 99.7pglml. The endogenous Leu-enkephalin level in untreated serum was 4Spg/ml. These data indicate that the recovery rate is rather high when Leu-enkephalin is added to serum and dialysis is not performed. Recovery of Leu-Enkephahn Exogenously added to Serum without Dialysis and without Actinonin Table 1
Added Leu-enkephalin
Concentration of Endogenous Leu-Enkephalin in Serum with or without Dialysis Table 2
Results
Measured
Recovered
Recovery
(pglml)
(pglml)
(pglml)
00)
0 11.1 99.7
4.5 13.5 86.7
9.0 82.2
81.1 82.4
The detected endogenous serum level of Leuenkephalin was increased from 4Spg/ml to 34.3pg/ml when the same serum was dialyzed against 25mM Tris/HCl buffer (pH 7.0) without addition of actinonin, as shown in Table 2. This experiment was repeated 7 times. Effect of actinonin addition and dialysis of serum Addition of actinonin to blood and dialysis of the
Leu-enkephalin
Experiment No.
(pglml)
without dialysis
with dialysis
1 2 3 4 5 6 7
4.7 3.6 5.4 3.7 4.0 3.4 6.7
40.9 36.7 30.1 28.8 30.9 37.9 36.1
Mean + SD
4.5 * 1.1
34.3 + 4.2
No actinonin was added to the serum.
serum increased the recovery of Leu-enkephalin as shown in Table 3. Leu-enkephalin was added to actinonin containing serum at a concentration of 50.4pg/ml followed by incubation at 4°C or 37°C for 3 hr. After that, the serum was dialyzed, and Leu-enkephalin was assayed by the same method as described above. The Leu-enkephalin recovery from the serum was 25.6% and 21.1% without addition of actinonin when it was incubated for 3 hr at 4°C or 37°C before dialysis respectively. On the other hand, the recovery rates were 90.3% and 89.9% when actinonin was added to the serum. There was no difference in the recovery rates with incubation at 4°C and 37°C. These data suggest that actinonin addition effectively prevented degradation of Leu-enkephalin in the serum during dialysis and increased the recovery rate from 25.6% to 90.3%.
Effect of Actinonin on Exogenously added Leu-Enkephalin Recovery at 4°C or 37°C Incubation in Serum before Dialysis
Table 3
Actinonin (Wglm!, 0
Incubation (3 hrs) 4°C 37°C 4°C
100 37°C
Added
Leu-enkephalin Measured
0
59.8
50.4 0 50.4 0
72.7 59.2 69.9 75.6 121.1 63.7 109.0
50.4 0 50.4
(pglml)
Recovery Recovered
(%) _
12.9
25.6
10.7
21.2
45.5
90.3
45.3
89.9
204
NEUROPEPTIDES
Serum Level of Leu-Enkephalin in Healthy Volunteers Table 4
Leu-enkephalin Actinonin
Dialysis
0kg/m1 lOOpg/ml
(+I (+I
0 t&ml
C-1
Leu-enkephalin
(P&4 21.1 93.4 1.0
level in serum of healthy volunteers
Table 4 shows the levels of Leu-enkephalin in the serum of the 12 healthy volunteers. The serum level of Leu-enkephalin was 93.4pgl ml when actinonin was added to blood to a concentration of lOO~g/ml. The serum Leu-enkephalin concentration without addition of actinonin was 2l.lpg/ml. Leu-enkephalin was 7.0pg/ml when it was assayed without actinonin and without dialysis. HPLC
analysis
The eluted sample was fractionated at 2ml/min and each of the fractions was assayed for Leuenkephalin concentration by the RIA method described above. The retention time of Leu-enkephalin from the samples was 17 min. Standard Leu-enkephalin had a retention time of 17 min in the same condition.
Discussion This paper describes a new method for detection of Leu-enkephalin in human serum by combination of the processes of dialysis and addition of an appropriate peptidase inhibitor, actinonin. Actinonin shows strong competitive inhibition with substrates for enkephalin-aminopeptidase, dipeptidyl aminopeptidase and enkephalinase A (20). Since human blood contains various peptidases, it was hypothesized that actinonin might protect Leu-enkephalin from degradation by these peptidases. On the other hand, dialysis is an efficient process for detecting Leu-enkephalin, as shown in Tables 1 and 2. The concentration in serum was increased 7.6 times by this process, as shown in Table 2.
The Leu-enkephalin plasma level in healthy persons was reported to be 54 f 10 (average + standard deviation) pg/ml by Ryder et al. (10) and below 3pg/ml by Yoshimasa et al. (12). However, we found the serum Leu-enkephalin level in 12 healthy volunteers using the present method to be 93.4 pg/ml, which is 1.73 times higher than Ryder et al’s value for 9 fasting healthy subjects (10). Both Ryder et al. and Yoshimasa et al. acidified the plasma to protect Leu-enkephalin from proteolytic enzymes, however, they assayed it by RIA methods without dialysis of the plasma. Their low value of the Leu-enkephalin level suggests that acidity failed to inactivate proteolytic enzymes completely or the enkephalin binding protein did not release Leu-enkephalin. Aloyo et al. (19) said that they could not completely inhibit Met-enkephalin breakdown with bestatin, an aminopeptidase inhibitor. They (19) suggested that bestatin did not inhibit aminopeptidase activity completely or that peptidases other than aminopeptidase were active in the blood and plasma. Thus, we felt that another better peptidase inhibitor might improve the detection of endogenous enkephalin-like substances. In our experiment, actinonin was used at a concentration of lOOl~,g/ml, which is 256 times more than its ICso for enkephalin-aminopeptidase, 91 times more than its ICSOfor dipeptidyl aminopeptidase and 18 times more than its IGO for enkephalinase A (20). The serum level of Leu-enkephalin was 93.4pg/ ml when assayed by our method, as shown in Table 4. On the contrary, the same serum showed 21.1 pg/ml when it was assayed after dialysis but without addition of actinonin. These data indicate that the combined process of addition of actinonin and dialysis is useful for detection of the Leu-enkephalin level in human serum. Our findings suggest that the difference between 93.4pg/ml with actinonin and 21.1 pg/ml without it is due to degradation by aminopeptidases in the serum during dialysis. Table 3 shows that pre-incubation temperatures of 4°C and 37°C gave equivalent results; therefore, it appears that exogenously-added Leu-enkephalin is not degraded in the serum during the incubation. Table 1 shows that exogenously-added Leu-enkephalin was recovered at rather high rates
205
ENDOGENOIJS ENKEPHALIN-DEGRADATION
from serum without either dialysis or actinonin, although the endogenous Leu-enkephalin concentration was as low as 4Spgfml. On the other hand, Table 3 shows that the recovery rate of exogenously-added Leu-enkephalin was reduced to about 25% by the process of dialysis when no actinonin was added to the serum. Addition of actinonin increased the recovery to about 90%. In the present method, actinonin seems to have protected Leu-enkephalin from aminopeptidases in the serum during dialysis for the long period of 24hr. We confirmed that the HPLC column retention time of Leu-enkephalin from the serum was identical with that of standard Leu-enkephalin. Since actinonin exerts strong competitive inhibitory activity against enkephalin-aminopepand dipeptidyl aminopeptidase tidase , enkephalinase A, it is hypothesized that addition of actinonin in serum will also be useful for detection of serum level of enkephalin-like substance other than Leu-enkephalin in man and animals. Use of the method presented here to accurately determine the serum level of opioid peptides in normal subjects and patients with various diseases will help elucidate the mechanisms and roles of opioid peptides in man and animals in the future.
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Acknowledgements The authors wish to express a special gratitude to Biomedical Laboratories for their assistance in the Leu-enkephalin assay.
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