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JCE & M • 1977 Vol 45 • No 4

COMMENTS

9. Mattingly, D., A simple fluorimetric method for estimating free 11-hydroxycorticoids in human plasmaj Clin Pathol 15: 374, 1962. 10. Ambrosi, B., P. Travaglini, P. Beck-Peccoz, R. Bara, R. Elli, A. Paracchi, and G. Faglia, Effect of sulpiride-induced hyperprolactinemia on serum testosterone response to HCG in normal men,/ Clin Endocrinol Metab 43: 700, 1976. 11. Rubin, R. T., R. E. Poland, and B. B. Tower, Prolactin-related testosterone secretion in normal adult man,/ Clin Endocrinol Metab 42: 112, 1976. 12. Bohnet, H. G., H. G. Dahlen, W. Wuttke, and H. P. G. Schneider, Hyperprolactinemic anovulatory syndrome,/ Clin Endocrinol Metab 42: 132, 1976.

13. Nader, S., K. Mashiter, F. H. Doyle, and G. F. Joplin, Galactorrhea, hyperprolactinemia and pituitary tumors in the female, Clin Endocrinol 5: 245,1976. 14. Vezina, J. L., and T. J. Sutton, Prolactin-secreting pituitary microadenomas, Am/ Roentgenol 120: 46, 1974. 15. Child, D. F., S. Nader, K. Mashiter, M. Kjeld, L. Banks, and T. R. Fraser, Prolactin studies in "functionless" pituitary tumours, Br MedJ 1: 604, 1975. 16. Magrini, G., J. R. Ebiner, P. Burckhardt, and J. P. Felber, Study on the relationship between plasma prolactin levels and androgen metabolism in man, J Clin Endocrinol Metab 43: 944, 1976. 17. Friesen, H. G. Prolactin and human reproduction, Res Reproduct 8: 3, 1976.

Serum Somatomedin Peptides Measured by Somatomedin A Radioreceptor Assay in Chronic Liver Disease KAZUE TAKANO, NAOMI HIZUKA, KAZUO SHIZUME, NAOAKI HAYASHI,* YOJI MOTOIKE,* AND HIROSHI OBATA* Department of Internal Medicine and * Institute of Gastroenterology, Tokyo Women's Medical College, Tokyo 162,Japan ABSTRACT. The levels of serum somatomedin peptides were determined with a somatomedin A radioreceptor assay utilizing human placental membranes. Low levels were found in 25 patients with liver cirrhosis and 28 patients with chronic hepatitis with the mean of 0.47 ± 0.05 and 0.60 ± 0.04 U/ml, respectively. There was a positive correlation be-

T

HERE are several reports which suggest that bioassayable somatomedin is released by liver (1-3). Wu et at. (4) and others (5-7) reported low levels of somatomedin activity determined by the bioassay in patients with chronic liver disease. This study reports serum somatomedin A determined by the radioreceptor assay in patients with liver cirrhosis and chronic hepatitis and its relationship to various liver function tests and serum growth hormone. The radioreceptor assay for somatomedin A not only measures the polypeptide somatomedin A but also, to some extent, biological related polypeptides such as somatomedin C (8). Received October 15, 1976. Supported in part by a research grant for "Specific Disease" of the Japanese Ministry of Health and Welfare and by a research grant of Yoshioka Memorial Fund of Tokyo Women's Medical College.

tween somatomedin A on one hand and serum albumin, cholinesterase, total cholesterol and thrombotest on the other. There was a negative correlation between somatomedin A and the indocyanine green retention test. These findings confirm earlier results obtained with bioassay. (/ Clin Endocrinol Metab 45: 828, 1977)

Materials and Methods Fifty-three patients with chronic liver disease were studied. Twenty-five patients, aged 26 to 73, had cirrhosis of the liver, which was confirmed by clinical, laboratory and pathological findings. The criteria for diagnosis were those adopted by the Congress of Havana (9). Twenty-eight patients, aged 26-62, were diagnosed as chronic hepatitis by liver biopsy according to the criteria of De Groote et al. (10). The etiology of the disease in these patients was unknown. Surface antigen or antibody of hepatitis B virus (HBsAg or HBsAb) were positive in 18 of 24 cases (75%) in liver cirrhosis and in 9 of 26 cases (35%) in chronic hepatitis. Further clinical and laboratory features are summarized in Tables 1 and 2. Serum concentration of somatomedin in healthy subjects was determined in 71 males and 60 females between the ages of 20 and 86 years. These normal subjects were selected from doctors, nurses and other employees working in our hospital. Venous blood samples were drawn after an overnight fast, centrifuged and the serum was stored at - 2 0 C

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829

COMMENTS TABLE 1. Clinical and laboratory data in patients with liver cirrhosis

Patient

1 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 X

SD SEM

Age

Sex

26 45 51 73 73 57 41 37 47 47 42 65 38 56 69 57 49 71 48 53 43 43 47 40 60

M M M M F F M M M M M M M F M F F F M M F M F M M

51.1 12.2 2.45

Alb g/dl

y-C g/dl

GOT KU

GPT KU

Alp KAU

ChE ApH

ICG %

T.Choi, mg/dl

T.T. %

3.0 2.7

196

88

36.3

3.7

2.2 2.7 1.6

39

24

10.8

3.0 3.2 3.7 3.5 3.4 4.1 3.7 3.6 4.4 4.0 3.7 4.1 3.1 3.2 1.9 3.7 4.0 2.6 3.0 2.9 1.8 3.9

2.1 1.2 2.4 1.8 1.0 1.5 2.7 1.1 1.6 2.4 1.4 2.1 3.0 2.1 2.4 1.4 2.4 1.9 2.2 3.0 2.0

137 26 46 35 79 31 92 29 30 66 23 35 134 50 64 29 123 66 47 30 56

34 29 28 44 91 30 52 15 19 64 15 15 49 23 41 14 49 40 22 15 35

10.2 7.6 15.0 8.0

0.43 0.19 0.77 0.29 0.17 0.31 0.49 0.58 1.12 0.98 0.79 1.02 0.81 0.59 0.91 0.41 0.64 0.19 0.70 0.44 0.38 0.54

40 59 29 36 37 34 34

165 95 156 136 101 143 113

78 28.5 44 64 51 98 100

3.36 0.65 0.13

2.01 0.57 0.12

63.6 44.9 9.4

36.3 21.8 4.5

until used. Serum somatomedin A was determined by the radioreceptor assay described earlier (11-13). The somatomedin A used for labelling was purified by Dr. L. Fryklund at Recip Polypeptide Laboratory, AB Kabi, Stockholm and was identical with that used in previous studies (13). In all cases a symmetrical 4-point design was used with two serum volumes of the local reference serum and of the unknown serum. Pooled serum from two healthy men and two healthy women between the ages of 20 and 25 years was used as an arbitrary laboratory standard. This reference serum pool was standardized by comparison with the level of serum somatomedin A in 24 normal subjects between the ages of 20 and 29 years, which was considered to be one unit of somatomedin A per ml. The mean precision (\ values) of all the results presented here is 0.10 ± 0.00 (SD 0.04) and intra- and interassay variation of repeated measurements on the same sample were 11.7% and 15.7%, respecitvely. Plasma GH was measured by a radioimmunoassay technique using dextran coated charcoal for separation of antibody bound and free hormone. Wilhelmi GH 51523D was used as standard. Standard laboratory methods were used to determine serum protein, cholinesterase (ChE), total cholesterol (T. Choi.), thrombo-

7.2 10.6 10.8 8.3 5.6 17.3 6.7 5.2 48.0 11.9 7.5 10.7 10.0 9.9

0.59 0.29 0.06

16 24

ng/ml

U/ml

1.5 14.9 1.3 8.2 32.9 3.7

2.6 1.3

0.44 0.12 0.25 0.28 0.26 0.31 0.27 0.58 0.64 0.64 0.75 0.74 0.91 0.67 1.12 0.87 0.31 0.25 0.37 0.49 0.28 0.49 0.22 0.10 0.42

4.93 7.35 1.64

0.47 0.26 0.05

1.7

1.0 1.3 1.1 2.5

28 25 21 31

4.9 1.2 3.1 2.8 5.5

29

31.6 10.2 2.7

Somatomedin A

2.4 4.7

0.24 1.05 12.8 10.6 2.4

GH

129.9 27.3 10.3

66.2 27.2 10.3

test (T. T.) (14), glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and alkaline phosphatase (Alp). Serum albumin concentrations were calculated by multiplying total protein by the percentage of albumin obtained with electrophoresis. Indocyanine green (ICG) test (15) which measures the excretion rate of ICG injected iv was also performed to estimate the liver cell function and the amount of blood circulating in the liver. Correlation coefficients between serum somatomedin A and other factors was calculated by the least squares method. Student's t test was applied when significances were determined.

Results All clinical and laboratory findings are listed in Tables 1 and 2. The levels of somatomedin A in serum from healthy controls at different ages ranging from 20 to 86 years did not change significantly (Table 3). Serum somatomedin A in 25 patients with liver cirrhosis varied between 0.10 and 1.12 U/ml with a mean of 0.47 ± 0.05 U/ml. In 28 patients with chronic hepatitis the values ranged from 0.33 to 1.33 U/ml with a mean of

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JCE & M • 1977 Vol 45 ( No 4

COMMENTS

830

TABLE 2. Clinical and laboratory data in patients with chronic hepatitis

Patient

Age

Sex

1 2 3 4 5 6 7 8 9 10 11 12 13 14

35 41 49 45 45 61 44 30 39 32 44 42 54 42 58 45

M M M M M M M M M M M M F M

15

16 17 18 19 20 21 22 23 24 25 26 27 28 X

SD SEM

26 39 49 62 47 26 55 48 58 39 45 50 44.6 9.6 1.8

M

M M F F F M F F M M F F M

Alb g/dl

y-G g/dl

GOT KU

GPT KU

Alp KAU

ChE ApH

ICG %

T.Choi. mg/dl

T.T. %

GH ng/ml

4.2 3.6 3.8 3.8 3.6 3.5 4.2 4.3 4.1 4.1 3.9 3.5

39 36 20

43 46 7

7.2 8.0 10.1

29 93 28 33 53 18 63 126 49 76

100 100 88 90 100 100

1.2 1.1 6.7 1.7 0.7 1.9

1.4 2.1

73 65

8.8 11.7 8.6 8.3 8.0 6.9 11.1 9.7 6.8 9.3 14.1

171 157 143 142 222 143

3.8 4.0

4.3 4.1 3.6 3.6 4.1 3.8 3.9 4.0 3.8 4.1 3.8 3.8

1.3 1.7 3.1 2.7 1.7 1.7 1.2 2.0 1.8 1.3 2.0 2.7

115 22 134 63 40 152 29 67 72 21 45 45

16 49 34 35 67 22 51 54 37 59 47 43 128 18 92 45 34 110 25 52 52 8 34 39

9.2 3.5 7.0 6.0 4.4 12.6 7.1 16.1 9.8 6.4 11.4 10.6

1.00 0.94 0.51 0.90 1.02 0.45 1.02 0.83 0.82 0.93 0.98 0.33 0.79 0.71 0.84 0.97 0.91 0.82 0.65 0.79 0.92 0.54 1.03 0.52 0.63 0.88 0.86 0.67

14 13 17 41 8 41

3.5 4.0

1.4 1.7 1.8 1.6 1.4 1.7 1.7 1.5 1.8 1.4 1.6 1.9 1.9 2.2

3.89 0.25 0.05

1.80 0.45 0.08

59.5 36.7 7.1

46.2 27.8 5.4

8.95 2.79 0.54

0.80 0.19 0.04

8.9

1.3 1.4 1.9 46.8 7 2.1 9 1.5

6

1.5

9

12.4 6.0 1.6 1.3 13.2

28 78

4.3

15

22.0 20.7 5.7

163.0 31.1 12.7

96.3 5.7 2.3

5.72 10.61 2.43

Somatomedin A U/ml 0.43 0.84 0.47 0.37 0.68 0.33 1.33 0.59 0.99 0.65 0.61 0.62 0.74 0.74 1.09 0.70 0.70 0.65 0.37 0.51 0.43 0.36 0.44 0.53 0.39 0.49 0.39 0.39 0.60 0.24 0.04

Normal levels of ChE are between 0.6 and 1.2. Fourteen patients (56%) with cirrhosis and 5 patients (17.9%) with chronic hepatitis showed ChE below normal. Thirteen patients (92%) out of 14 with cirrhosis and 4 patients (31%) out of 13 with chronic hepatitis had more than 20% retention of ICG 15 minutes after injection. Serum cholesterol and thrombotest were performed in a small number of cases. Their values were lower in patients with liver cirrhosis than in those with 3. Somatomedin A levels in normal subjects chronic hepatitis. SomatoThe somatomedin results in patients with liver Number medin A cirrhosis and chronic hepatitis were correlated (U/ml) with biochemical findings and results of liver Mean ± SD Total Male Female function tests. As illustrated in Table 4, a positive 24 12 12 1.00 ± 0.23 correlation was found between somatomedin A 25 13 12 0.89 ± 0.29 on one hand and serum albumin, ChE, total 31 17 14 0.85 ± 0.31 cholesterol and thrombotest on the other. There 12 30 18 0.83 ± 0.36 was a negative correlation between the serum 11 21 10 0.80 ± 0.26 level of somatomedin A and the ICG retention

0.60 ± 0.04 U/ml. These values were significantly lower than those obtained in normal subjects (P < 0.005). Levels of somatomedin A in patients with liver cirrhosis were lower than those in chronic hepatitis (0.05 < P < 0.1). Serum albumin concentrations at or below 3.0 g/dl were found in 8 patients (32%) with liver cirrhosis and in none (0%) with chronic hepatitis. TABLE

Age 20-29 30-39 40-49 50-59 60-86

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831

TABLE 4. Correlation coefficients between serum somatomedin A (Sm A) and other factors in liver disease Liver cirrhosis

Combined

Sm A vs. albumin Sm A vs. ChE Sm A vs. ICG Sm A vs. In hGH Sm A vs. T. T. Sm A vs. T. cholesterol

Chronic hepatitis

n

r

n

r

n

r

53 52 23 39 13 13

0.53* 0.53* -0.50| -0.36| 0.61| 0.701

25 24 12 20 7 7

0.621 0.60* -0.68$ -0.47$ 0.61 0.74

28 28 11 19 6 6

0.24 0.35 -0.43 -0.26 0.37 0.48

* P < 0.005; f P < 0.01; t P < 0.05. n = number of cases; r = correlation coefficient.

test and the level of growth hormone. Separate analysis of these correlations in patients with cirrhosis and with chronic hepatitis disclosed that significant correlations were found only in patients with cirrhosis. There was no correlation between the level of somatomedin A on one hand and GOT, GPT and Alp on the other.

would result in lower levels of total serum somatomedin. However, no study concerning the amount of somatomedin binding protein in liver diseases has yet been reported. Other factors such as nutritional deficiency and/or metabolic changes induced by the dysfunction of the liver might be contributing to the low levels of serum somatomedin.

Discussion The levels of serum somatomedin peptides were determined with a somatomedin A radioreceptor assay. Low levels of somatomedin A were found in patients both with liver cirrhosis and with chronic hepatitis. These results are similar to those observed by bioassay (4-6) and support the hypothesis that somatomedin is generated by the liver (1-3,16,17). As shown in Table 4, in chronic liver disease there were good correlations between reduced levels of somatomedin A and decreased levels of ablumin, ChE, prothrombin and total cholesterol, all of which are produced by the liver. The results of the ICG test at 15 min after injection also had a negative correlation with serum somatomedin A. However, significant correlations with these laboratory tests were found only in patients with liver cirrhosis. This finding is probably the result of the advanced impairment of liver function. Because of these results we think that the generation of somatomedin A is reduced earlier than that of other substances such as albumin and ChE. The existence of a binding protein for somatomedin in serum is amply confirmed (18,19), although this protein has not been well characterized. Most of the somatomedin in serum is bound to the binding protein. Since our assay measures both protein bound and free somatomedin (20), a reduced level of binding protein

Acknowledgments The authors are greatly indebted to Doctors Linda Fryklund and Hans Sievertsson from the Research Department of AB KABI, Stockholm, who supplied us with the somatomedin A preparations. We want to thank Miss Tomoko Katagiri for her technical assistance.

References 1. McConaghey, P., and L. B. Sledge, Production of "sulphation factor" by the perfused liver, Nature (Lond) 225: 1249, 1970. 2. Hall, K., and K. Uthne, Some biological properties of purified sulfation factor (SF) from human plasma, Ada Med Scand 190: 137, 1971. 3. Schimpff, R. M., M. Donnadieu, J. G. Glasinovic, J. M. Warnet, and F. Girard, The liver as a source of somatomedin, Ada Endocrinol (Kbh) 83: 365, 1976. 4. Wu, A., D. B. Grant, J. Hambley, and A. J. Levi, Reduced serum somatomedin activity in patients with chronic liver disease, Clin Sci Mol Med 47: 359, 1974. 5. Wiedemann, E., E. Schwarts, A. Purohit, S. Valencia, and J. Sanchez, Serum somatomedin activity in chronic liver disease, Clin Res 22: 483A, 1974. 6. Bala, R. M., C. Hankins, and G. R. Smith, A somatomedin assay using normal rabbit cartilage in clinical studies, Can] Physiol Pharmacol 53: 403,1975. 7. Marek, J., M. Schullerova, and O. Schreiberova, Somatomedin in chronic liver disease, Rev Czech Med 22: 194, 1976.

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832

COMMENTS

8. Hall, K., K. Takano, L. Fryklund, and H. Sievertsson, The measurement of somatomedin A by radioreceptor assay, Adv Metab Disord 8: 61, 1975. 9. Scherlock, S., B. Sepulveda, P. A. Castillo, L. Schiff, H. Popper, and A. J. French, Report of the board for classification and nomenclature of cirrhosis of the liver, Gastroenterology 31: 213, 1956. 10. De Groote, J., V. J. Desmet, P. Gedigk, G. Korb, H. Popper, H. Poulsen, P. J. Scheuer, M. Schmid, H. Thaler, and E. Uehlinger, A classification of chronic hepatitis, Lancet 2: 626, 1968. 11. Hall, K., K. Takano, and L. Fryklund, Radioreceptor assay for somatomedin A,/ Clin Endocrinol Metab 39: 973, 1974. 12. Takano, K., K. Hall, L. Fryklund, A. Holmgren, H. Sievertsson, and K. Uthne, The binding of insulin and somatomedin A to human placental membrane, Ada Endocrinol (Kbh) 80: 14, 1975. 13. Takano, K., K. Hall, M. Ritzen, L. Iselius, and H. Sievertsson, Somatomedin A in human serum, determined by radioreceptor assay, Ada Endocrinol (Kbh) 82: 449, 1976. 14. Owren, P. A., Thrombotest, a new method for controlling anticoagulant therapy, Lancet 2: 754, 1959.

JCE & M • 1977 Vol 45 • No 4

15. Hunton, D. B., J. L. Bollman, and H. N. Hoffman II, Studies of hepatic function with indocyanine green, Gastroenterology 39: 713, 1960. 16. Phillips, L. S., A. C. Herington, I. E. Karl, and W. H. Daughaday, Comparison of somatomedin activity in perfusates of normal and hypophysectomized rat livers with and without added growth hormone, Endocrinology 98: 606, 1976. 17. Uthne, K., and T. Uthne, Influence of liver resection and regeneration on somatomedin (sulfation factor) activity in sera from normal and hypophysectomized rats, Ada Endocrinol (Kbh) 71: 255, 1972. 18. Hall, K., Human somatomedin, Ada Endocrinol [Suppl] (Kbh) 163: 1, 1972. 19. Hintz, R. L., E. M. Orsini, and M. G. Van Camp, Evidence for a somatomedin binding protein in plasma, The Endocrine Society 56 Annual Meeting Program, 1974, A-71. 20. Hall, K., K. Takano, G. Enberg, and L. Fryklund, Studies on the regulation of somatomedins A and B, In Pecile, A., and E. E. Miiller (eds.), Growth Hormone and Related Peptides, ICS series No. 381, Excerpta Medica, Amsterdam, 1976, p. 178.

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Serum somatomedin peptides measured by somatomedin A radioreceptor assay in chronic liver disease.

828 JCE & M • 1977 Vol 45 • No 4 COMMENTS 9. Mattingly, D., A simple fluorimetric method for estimating free 11-hydroxycorticoids in human plasmaj...
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