Permeability of Antidiuretic Hormone and Other Hormones Through the Dialysis Membrane in Patients Undergoing Chronic Hemodialysis KAZUAKI SHIMAMOTO, TOSHIAKI ANDO, TAKASHI NAKAO, IKUO WATARAI,* AND MITSUO MIYAHARA Second Department of Internal Medicine, Sapporo Medical College, S-l W-16, Sapporo, Japan, and *Watarai Iin Clinic, S-14 W-15, Sapporo, Japan ABSTRACT. In eight patients undergoing chronic hemodialysis, ultrafiltration was performed for 1 h in each patient. The concentration of urea nitrogen, creatinine, ADH, cortisol, GH, prolactin and TSH was measured in plasma and the filtering solution, and the permeability of each substance was determined. The plasma concentration of ADH coincided with that of the filtering solution, and no significant difference was noted between the permeability of creatinine and ADH. In contrast, cortisol, GH,
R
ECENTLY studies on ADH release in patients undergoing chronic hemodialysis were reported by us (1) and Pastoriza-Munoz et al. (2). However, whether ADH passes through the dialysis membrane or whether ADH binds to plasma proteins has been controversial in the experiments of both man (3-6) and animals (6). In this study, therefore, the permeability of ADH and other hormones such as cortisol, GH, prolactin and TSH through the dialysis membrane was investigated in order to clarify the permeability of these hormones and the blood transport pattern of ADH, in patients undergoing chronic hemodialysis, using extracorporeal ultrafiltration methods (ECUM). Materials and Methods
The experiment was performed in eight patients undergoing chronic hemodialysis ranging in age from 24 to 51 years. The nature of the renal disease was chronic glommerulonephritis (6 cases), polycystic kidney (1 case) and a single case of unknown renal disease. Ultrafiltration was performed for 1 h in each case. Blood samples were drawn immediately before and after the ultrafiltration and the filtering solution was collected for measurement of ADH, prolactin, GH, cortisol, TSH, urea nitrogen (UN) and creatinine. The permeability of each substance through the dialysis membrane was shown for the clearance of ultrafiltration and was calculated by the following formula, permeability = 2 x FvFc/(Pbc + Pac) ml/ min, where Fv and Fc are the volume (ml/min) and concentration of the substance of the filtering solution, respectively, and Pbc and Pac are the plasma Received November 30, 1976.
prolactin and TSH were not detected in the filtering solution. Chromatographic study showed that ADH in the filtering solution coincided with synthetic ADH. From a comparison of the permeability with the molecular weight, it was suggested that ADH in the blood exists in free form without binding with plasma proteins or neurophysin. (J Clin Endocrinol Metah 45: 818, 1977)
concentration of the substance before and after ultrafiltration, respectively. Extracorporeal ultrafiltration methods (ECUM) This is a method of ultrafiltration without using dialysate solution. All patients were dialyzed by the EX-23 DIALYZER CARTRIDGE (Extra-corporeal Medical Specialties, Inc., U.S.A.), and ultrafiltration pressure was 200 mmHg. Chromatographic study ADH was extracted from the filtering solution by the same method used in plasma samples, and this extracted solution and synthetic ADH (Sigma Chemical Company, USA) was applied to 1.0 by 25 cm column chromatography of fine Sephadex G-25 and eluted with 0.05M phosphate buffer (pH 7.4). Each 1 ml fraction was collected and measured for ADH. UN and creatinine were measured by an autoanalyzer. ADH levels were detected by the heterologous radioimmunoassay method previously reported (7). Cortisol, GH, prolactin and TSH were measured by radioimmunoassay methods. Statistical analysis was performed with Student's t test for paired data.
Results Collected filtering volume was 552 ± 34 ml (mean ± SEM). Plasma concentration before (Pbc) and after (Pac) ultrafiltration, the concentration in filtering solution (Fc), and the permeability of urea, creatinine, ADH, cortisol, GH, prolactin and TSH are shown in Table 1. There was no significant difference between Pbc and Pac of UN, creatinine, ADH, cortisol, GH, prolac-
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COMMENTS
819
TABLE 1. The permeability of urea, creatinine, ADH, cortisol, GH, prolactin (hPr) and TSH Case
ing/dl
Creatinine mg/dl
59.3 62.2 63.7
12.2 12.7 12.4
7.1 5.8
8.7
8.3
7.4
74.3 77.7 75.5 11.1
13.3 14.1 12.9 10.5
10.8 11.3
93.5 99.3 100.7
14.0 14.8 14.6
7.0
6.8
Perm
94.7 92.2 95.5 11.5
20.6 20.2 20.3 11.2
Pbc Pac Fc
74.0 76.0 75.2
16.5 16.5 16.5
8.7
8.7
75.0 76.0 73.3 10.0
17.2 17.5 16.9 10.0
7.8 6.3
62.0 63.0 62.0 8.4
13.0 12.5 12.2 8.1
7.1 6.7 6.0 7.4
11.5 11.0
66.0 67.0 70.5
13.0 13.0 13.6
5.2
5.2 7.4
9.2
9.1
UN
No.
Age
Sex
1
51
F
Pbc Pac Fc
Perm 2
34
M
Pbc Pac Fc
Perm 3
51
M
Pbc Pac Fc
Perm 4
40
M
Pbc
Pac Fc 5
24
M
Perm 6
30
M
Pbc Pac Fc
Perm 7
44
F
Pbc Pac Fc
Perm 8
46
F
Pbc Pac Fc
Perm Mean ± SEM
Pbc Pac Fc
Perm
74.9 ± 76.8 ± 77.1 ± 9.3 ±
4.7 4.7 4.9 0.5
15.0 ± 15.2 ± 14.9 ± 9.1 ±
1.0 1.0 1.0 0.5
ADH
pg/ml
5.8
Cortisol fig/dl 9.6 5.8 0 0
hPr ng/ml
GH
ng/ml 10.1
16.7 16.0
0 0
0 0
6.8
5.5 3.0 0 0
2.1 2.2 0 0
26.0 27.8 0
8.2 4.7 6.7 6.9
11.8 11.6
8.6 3.1 0
33.0 32.0
7.4 4.1 5.7
2.7 2.5 0 0
18.6 19.0
11.1
6.2 2.9 0 0
5.8 5.6 5.6 8.5
6.5 9.2 0 0
— — —
—
6.3
5.8 5.0 0 0
—
—
—
—
— — —
—
8.1 8.2
9.1
4.6 5.2 9.2
7.2 6.3 6.2 8.5
± ± ± ±
0.6 0.8 0.3 0.5
0 0
0 0
0
0 7.8 ± 1.0 7.0 ± 1.2 0 0
0
0 0
0
0 0
—
—
TSJ /u,U/ml 9.2 7.8 0 0 6.6 8.2 0 0 19.0 15.2 0 0
— 3.1 3.5 0 — 3.8 3.6 0 0 3.0 3.0 0 0
10.8 10.5 — —
— —
0 0
5.1 ± 1.6 4.5 ± 1.9 0 0
23.6 ±3.7 23.7 ± 3.7 0 0
7.9 ± 2.2 7.4 ± 1.7 0 0
Pbc: Plasma concentration before ultrafiltration; Pac: Plasma concentration after ultrafiltration; Fc: Filtering solution concentration; Perm: Permeability of each substance (ml/min).
tin and TSH. In addition, UN, creatinine and ADH showed no significant difference between the plasma concentration and Fc. However, cortisol, GH, prolactin and TSH were not detected in the filtering solution. The mean permeability value was larger in the order of urea, creatinine and ADH. Despite a significant difference in permeability between urea and creatinine or ADH, no significant difference in permeability was found between creatinine and ADH. Chromatographically the elution pattern of ADH in the filtering solution showed one peak and this peak coincided with the peak of synthetic ADH (Fig. 1).
Discussion The present study showed that no significant difference between plasma levels and Fc was recognized in UN, creatinine and ADH, and that the permeability of ADH through the dialysis membrane was approximately consistent with that of urea and creatinine which are known to be completely dialyzable. Chromatographically it was shown that the ADH detected in the filtering solution was intact. In contrast, cortisol which is lower than ADH in molecular weight, was not detected in the filtering solution. Assuming that ADH, as in the case of cortisol,
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without binding with plasma proteins or neurophysin. 40
Acknowledgment The authors are indebted to Miss K. Abe for her technical assistance.
References
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Fraction number
FIG. 1. Fractionation of synthetic ADH (O
O) and
the extract solution of filtering solution ( • • ) on a column chromatography (1 x 25 cm) of Sephadex G-25 fine in 0.05M phosphate buffer (pH 7.4).
binds with plasma protein of high molecular weight, Fc of ADH should be lower than the plasma levels of ADH and the permeability of ADH should be lower than that of urea and creatinine. It is suggested, therefore, that ADH does not bind with a plasma protein which has a high molecular weight. It is well known that neurophysin is a ADHbinding polypeptide which exists in human blood (8-10). Recendy, Kazin et al. reported that plasma levels of ADH and neurophysin rise simultaneously during smoking (11). Therefore, it is possible that ADH exists in bound form with neurophysin in the blood. Although the molecular weight of neurophysin is not known exactly, it has been estimated to be about 10,000 or 20,000 (8,9,12). If its molecular weight is about 20,000 as Hollenberg and Hope reported (12), it is approximately consistent with that of GH, prolactin and TSH, which were not detected in the filtering solution. On the other hand, if it is about 10,000 as reported by Cheng et al. (8), it is approximately the same as the molecular weight of /32microglobulin. It has been shown that the permeability of /32-microglobulin is very small and its clearance is 3.0% of urea clearance on ultrafiltration (13). Therefore, ADH may exist either in unbound form as loosely bound to the neurophysin in blood. Thus, our study suggests that ADH in blood exists in free form, at least in uremic patients,
1. K. Shimanoto, I. Watarai, and M. Miyahara, A study of plasma vasopressin in patients undergoing chronic hemodialysis, J Clin Endocrinol Metab 45: 714, 1977. 2. Pastoriza-Munoz, E., R. E. Eastering, and R. L. Malvin, The effect of plasma calcium on plasma ADH levels in anephric patients, Nephron 16: 449,1976. 3. Czaczkes, J. W., C. R. Kleeman, and Koenig, M., Physiologic studies of antidiuretic hormone by its
4.
5.
6. 7.
8.
9.
10.
11.
12.
13.
direct measurement in human plasma,/ Clin Invest 43: 1625, 1964. Baumann, G., and J. F. Dingman, Distribution, blood transport, and degradation of antidiuretic hormone in man,/ Clin Invest 57: 1109, 1976. Ahmed, A. B. J., B. C. Jeorge, G. GonzalezAuvert, and J. F. Dingman, Increased arginine vasopressin in clinical adrenocortical insufficiency and its inhibition by glucosteroid, / Clin Invest 46: 111, 1967. Lauson, H. D., Metabolism of antidiuretic hormones, A m / Med 42: 713, 1967. Shimamoto, K., T. Murase, and T. Yamaji, The heterologous radioimmunoassay for arginine vasopressin,/ Lab Clin Med 87: 338, 1976. Cheng, K. W., and H. G. Friesen, The isolation and characterization of human neurophysin, / Clin Endocrinol Metab 34: 165, 1972. Robinson, A. G., and A. G. Franz> Radioimmunoassay of posterior pituitary peptide: A review, Metabolism 22: 1047, 1973. Robinson, A. G., Isolation, assay, and secretion of individual human neurophysins,/ Clin Invest 55: 360, 1975. Husain, M. K., A. G. Franz, F. Ciarochi, and A. G. Robinson, Nicotine stimulated release of neurophysin and vasopressin in humans, / Clin Endocrinol Metab 41: 1113, 1975. Hollenberg, M. D., and D. B. Hope, The isolation of the native hormone-binding proteins from bovine pituitary posterior lobes: crystallization of neurophysin-1 and -1 as complexes with (8arginine)-vasopressin, Biochem J 106: 557, 1968. E. Yokoyama, S. Furuya, Y. Kumamoto, K. Sugawara, and E. Chiba, Kinetics and permeability through the dialysis membrane of /32-microgloblin in chronic hemodialysis, Jap J Nephrol (In press).
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R
ECENTLY studies on ADH release in patients undergoing chronic hemodialysis were reported by us (1) and Pastoriza-Munoz et al. (2). However, whether ADH passes through the dialysis membrane or whether ADH binds to plasma proteins has been controversial in the experiments of both man (3-6) and animals (6). In this study, therefore, the permeability of ADH and other hormones such as cortisol, GH, prolactin and TSH through the dialysis membrane was investigated in order to clarify the permeability of these hormones and the blood transport pattern of ADH, in patients undergoing chronic hemodialysis, using extracorporeal ultrafiltration methods (ECUM). Materials and Methods
The experiment was performed in eight patients undergoing chronic hemodialysis ranging in age from 24 to 51 years. The nature of the renal disease was chronic glommerulonephritis (6 cases), polycystic kidney (1 case) and a single case of unknown renal disease. Ultrafiltration was performed for 1 h in each case. Blood samples were drawn immediately before and after the ultrafiltration and the filtering solution was collected for measurement of ADH, prolactin, GH, cortisol, TSH, urea nitrogen (UN) and creatinine. The permeability of each substance through the dialysis membrane was shown for the clearance of ultrafiltration and was calculated by the following formula, permeability = 2 x FvFc/(Pbc + Pac) ml/ min, where Fv and Fc are the volume (ml/min) and concentration of the substance of the filtering solution, respectively, and Pbc and Pac are the plasma Received November 30, 1976.
prolactin and TSH were not detected in the filtering solution. Chromatographic study showed that ADH in the filtering solution coincided with synthetic ADH. From a comparison of the permeability with the molecular weight, it was suggested that ADH in the blood exists in free form without binding with plasma proteins or neurophysin. (J Clin Endocrinol Metah 45: 818, 1977)
concentration of the substance before and after ultrafiltration, respectively. Extracorporeal ultrafiltration methods (ECUM) This is a method of ultrafiltration without using dialysate solution. All patients were dialyzed by the EX-23 DIALYZER CARTRIDGE (Extra-corporeal Medical Specialties, Inc., U.S.A.), and ultrafiltration pressure was 200 mmHg. Chromatographic study ADH was extracted from the filtering solution by the same method used in plasma samples, and this extracted solution and synthetic ADH (Sigma Chemical Company, USA) was applied to 1.0 by 25 cm column chromatography of fine Sephadex G-25 and eluted with 0.05M phosphate buffer (pH 7.4). Each 1 ml fraction was collected and measured for ADH. UN and creatinine were measured by an autoanalyzer. ADH levels were detected by the heterologous radioimmunoassay method previously reported (7). Cortisol, GH, prolactin and TSH were measured by radioimmunoassay methods. Statistical analysis was performed with Student's t test for paired data.
Results Collected filtering volume was 552 ± 34 ml (mean ± SEM). Plasma concentration before (Pbc) and after (Pac) ultrafiltration, the concentration in filtering solution (Fc), and the permeability of urea, creatinine, ADH, cortisol, GH, prolactin and TSH are shown in Table 1. There was no significant difference between Pbc and Pac of UN, creatinine, ADH, cortisol, GH, prolac-
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COMMENTS
819
TABLE 1. The permeability of urea, creatinine, ADH, cortisol, GH, prolactin (hPr) and TSH Case
ing/dl
Creatinine mg/dl
59.3 62.2 63.7
12.2 12.7 12.4
7.1 5.8
8.7
8.3
7.4
74.3 77.7 75.5 11.1
13.3 14.1 12.9 10.5
10.8 11.3
93.5 99.3 100.7
14.0 14.8 14.6
7.0
6.8
Perm
94.7 92.2 95.5 11.5
20.6 20.2 20.3 11.2
Pbc Pac Fc
74.0 76.0 75.2
16.5 16.5 16.5
8.7
8.7
75.0 76.0 73.3 10.0
17.2 17.5 16.9 10.0
7.8 6.3
62.0 63.0 62.0 8.4
13.0 12.5 12.2 8.1
7.1 6.7 6.0 7.4
11.5 11.0
66.0 67.0 70.5
13.0 13.0 13.6
5.2
5.2 7.4
9.2
9.1
UN
No.
Age
Sex
1
51
F
Pbc Pac Fc
Perm 2
34
M
Pbc Pac Fc
Perm 3
51
M
Pbc Pac Fc
Perm 4
40
M
Pbc
Pac Fc 5
24
M
Perm 6
30
M
Pbc Pac Fc
Perm 7
44
F
Pbc Pac Fc
Perm 8
46
F
Pbc Pac Fc
Perm Mean ± SEM
Pbc Pac Fc
Perm
74.9 ± 76.8 ± 77.1 ± 9.3 ±
4.7 4.7 4.9 0.5
15.0 ± 15.2 ± 14.9 ± 9.1 ±
1.0 1.0 1.0 0.5
ADH
pg/ml
5.8
Cortisol fig/dl 9.6 5.8 0 0
hPr ng/ml
GH
ng/ml 10.1
16.7 16.0
0 0
0 0
6.8
5.5 3.0 0 0
2.1 2.2 0 0
26.0 27.8 0
8.2 4.7 6.7 6.9
11.8 11.6
8.6 3.1 0
33.0 32.0
7.4 4.1 5.7
2.7 2.5 0 0
18.6 19.0
11.1
6.2 2.9 0 0
5.8 5.6 5.6 8.5
6.5 9.2 0 0
— — —
—
6.3
5.8 5.0 0 0
—
—
—
—
— — —
—
8.1 8.2
9.1
4.6 5.2 9.2
7.2 6.3 6.2 8.5
± ± ± ±
0.6 0.8 0.3 0.5
0 0
0 0
0
0 7.8 ± 1.0 7.0 ± 1.2 0 0
0
0 0
0
0 0
—
—
TSJ /u,U/ml 9.2 7.8 0 0 6.6 8.2 0 0 19.0 15.2 0 0
— 3.1 3.5 0 — 3.8 3.6 0 0 3.0 3.0 0 0
10.8 10.5 — —
— —
0 0
5.1 ± 1.6 4.5 ± 1.9 0 0
23.6 ±3.7 23.7 ± 3.7 0 0
7.9 ± 2.2 7.4 ± 1.7 0 0
Pbc: Plasma concentration before ultrafiltration; Pac: Plasma concentration after ultrafiltration; Fc: Filtering solution concentration; Perm: Permeability of each substance (ml/min).
tin and TSH. In addition, UN, creatinine and ADH showed no significant difference between the plasma concentration and Fc. However, cortisol, GH, prolactin and TSH were not detected in the filtering solution. The mean permeability value was larger in the order of urea, creatinine and ADH. Despite a significant difference in permeability between urea and creatinine or ADH, no significant difference in permeability was found between creatinine and ADH. Chromatographically the elution pattern of ADH in the filtering solution showed one peak and this peak coincided with the peak of synthetic ADH (Fig. 1).
Discussion The present study showed that no significant difference between plasma levels and Fc was recognized in UN, creatinine and ADH, and that the permeability of ADH through the dialysis membrane was approximately consistent with that of urea and creatinine which are known to be completely dialyzable. Chromatographically it was shown that the ADH detected in the filtering solution was intact. In contrast, cortisol which is lower than ADH in molecular weight, was not detected in the filtering solution. Assuming that ADH, as in the case of cortisol,
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JCE & M • 1977 Vol 45 • No 4
COMMENTS
820
without binding with plasma proteins or neurophysin. 40
Acknowledgment The authors are indebted to Miss K. Abe for her technical assistance.
References
0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Fraction number
FIG. 1. Fractionation of synthetic ADH (O
O) and
the extract solution of filtering solution ( • • ) on a column chromatography (1 x 25 cm) of Sephadex G-25 fine in 0.05M phosphate buffer (pH 7.4).
binds with plasma protein of high molecular weight, Fc of ADH should be lower than the plasma levels of ADH and the permeability of ADH should be lower than that of urea and creatinine. It is suggested, therefore, that ADH does not bind with a plasma protein which has a high molecular weight. It is well known that neurophysin is a ADHbinding polypeptide which exists in human blood (8-10). Recendy, Kazin et al. reported that plasma levels of ADH and neurophysin rise simultaneously during smoking (11). Therefore, it is possible that ADH exists in bound form with neurophysin in the blood. Although the molecular weight of neurophysin is not known exactly, it has been estimated to be about 10,000 or 20,000 (8,9,12). If its molecular weight is about 20,000 as Hollenberg and Hope reported (12), it is approximately consistent with that of GH, prolactin and TSH, which were not detected in the filtering solution. On the other hand, if it is about 10,000 as reported by Cheng et al. (8), it is approximately the same as the molecular weight of /32microglobulin. It has been shown that the permeability of /32-microglobulin is very small and its clearance is 3.0% of urea clearance on ultrafiltration (13). Therefore, ADH may exist either in unbound form as loosely bound to the neurophysin in blood. Thus, our study suggests that ADH in blood exists in free form, at least in uremic patients,
1. K. Shimanoto, I. Watarai, and M. Miyahara, A study of plasma vasopressin in patients undergoing chronic hemodialysis, J Clin Endocrinol Metab 45: 714, 1977. 2. Pastoriza-Munoz, E., R. E. Eastering, and R. L. Malvin, The effect of plasma calcium on plasma ADH levels in anephric patients, Nephron 16: 449,1976. 3. Czaczkes, J. W., C. R. Kleeman, and Koenig, M., Physiologic studies of antidiuretic hormone by its
4.
5.
6. 7.
8.
9.
10.
11.
12.
13.
direct measurement in human plasma,/ Clin Invest 43: 1625, 1964. Baumann, G., and J. F. Dingman, Distribution, blood transport, and degradation of antidiuretic hormone in man,/ Clin Invest 57: 1109, 1976. Ahmed, A. B. J., B. C. Jeorge, G. GonzalezAuvert, and J. F. Dingman, Increased arginine vasopressin in clinical adrenocortical insufficiency and its inhibition by glucosteroid, / Clin Invest 46: 111, 1967. Lauson, H. D., Metabolism of antidiuretic hormones, A m / Med 42: 713, 1967. Shimamoto, K., T. Murase, and T. Yamaji, The heterologous radioimmunoassay for arginine vasopressin,/ Lab Clin Med 87: 338, 1976. Cheng, K. W., and H. G. Friesen, The isolation and characterization of human neurophysin, / Clin Endocrinol Metab 34: 165, 1972. Robinson, A. G., and A. G. Franz> Radioimmunoassay of posterior pituitary peptide: A review, Metabolism 22: 1047, 1973. Robinson, A. G., Isolation, assay, and secretion of individual human neurophysins,/ Clin Invest 55: 360, 1975. Husain, M. K., A. G. Franz, F. Ciarochi, and A. G. Robinson, Nicotine stimulated release of neurophysin and vasopressin in humans, / Clin Endocrinol Metab 41: 1113, 1975. Hollenberg, M. D., and D. B. Hope, The isolation of the native hormone-binding proteins from bovine pituitary posterior lobes: crystallization of neurophysin-1 and -1 as complexes with (8arginine)-vasopressin, Biochem J 106: 557, 1968. E. Yokoyama, S. Furuya, Y. Kumamoto, K. Sugawara, and E. Chiba, Kinetics and permeability through the dialysis membrane of /32-microgloblin in chronic hemodialysis, Jap J Nephrol (In press).
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