0021-972X/91/7304-0777$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright (P) 1991 by The Endocrine Society
Vol. 73, No. 4 Printed in U.S.A.
Decreased Serum Growth Hormone-Binding Protein in Patients with Liver Cirrhosis YAACOV BARUCH, TAMAR AMIT, PNINA HERTZ, RAFAEL ENAT, MOUSSA B. H. YOUDIM, AND ZEEV HOCHBERG Department of Medicine B, Rambam Medical Center (Y.B., R.E.), and the Rappaport Family Institute for Research in the Medical Sciences and Departments of Medicine (Y.B., R.E.) and Pharmacology (T.A., P.H., M.B.H. Y., Z.H.), Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096 Haifa, Israel
ABSTRACT. The recently characterized GH-binding protein (GH-BP) has an amino acid sequence identical to the extracellular domain of the GH receptor. Serum GH-BP reflects the amount of GH receptors, and the liver seems to be their main source. To evaluate the effect of liver disease on GH-BP, 52 patients with liver cirrhosis were studied. Serum GH-BP was measured by a binding assay with dextran-coated charcoal separation. Levels of GH-BP were correlated against the clinical state, assessed by Pugh's score. The GH-BP of 31 Pugh's class A patients was 9.7 ± 0.5%/50 ML serum, and that of 21 Pugh's class B and C patients was 7.2 ± 0.5%/50 nh serum compared
to 11.3 ± 0.5%/50 jiL serum in age-matched controls. GH-BP correlated negatively with Pugh's score and serum bilirubin, and positively with serum albumin. It did not correlate with serum liver enzymes or serum insulin-like growth factor-I. Scatchard analysis of GH binding to the GH-BP revealed similar binding affinities in Pugh's A, B, and C patients and controls. The binding capacity in cirrhosis was significantly lower than that in controls. We conclude that serum GH-BP is controlled mainly by the liver and can provide an additional measure of disease severity in liver cirrhosis. (J Clin Endocrinol Metab 73: 777780,1991)
A
found in 27 of these patients. Based on both clinical and biochemical parameters, they were classified by Pugh's score (6). Pugh's score is composed of five variables. Two are assessed clinically (severity of ascites and encephalopathy), and three are based on laboratory tests (serum albumin and bilirubin levels and prothrombin time). Each variable is given a value of 1, 2, or 3 for increasing abnormality, and the values of the five variables are summed for each patient. Thus, the best possible score is 5, and the worst is 15. Using a continuous scale from 5-15, we get a finely graded measurement of liver disease severity. For simplicity, it can be presented as three classes: class A, 5-6; class B, 7-10; and class C, 11-15. This score has recently been validated as a predictor of prognosis in liver cirrhosis that is correlated with normal functional liver mass (7). Twelve age-matched healthy volunteers (8 males and 4 females) with normal serum liver enzyme tests, protein levels, and prothrombin time served as controls. All patients and volunteers consented to participate in the study. Diabetes mellitus was diagnosed in 12 patients with fasting blood glucose levels above 8.3 mmol/L.
SPECIFIC GH-binding protein (GH-BP) has recently been characterized in human serum (1, 2). A similar protein in rabbit serum has been found to have an amino acid sequence identical to the extracellular domain of the GH receptor (3). The liver may be an important source of GH-BP, and its serum levels have been shown to be reduced in a small number of patients with cirrhosis of the liver (4, 5). The aim of this work was to evaluate in a large group of patients the impact of reduced functional liver mass on GH-BP and to probe the possible role of GH-BP in the evaluation of the severity of liver cirrhosis.
Subjects and Methods Fifty-two patients (27 males and 25 females) with nonalcoholic liver cirrhosis were included in this study. The mean ages of the men and women were 58 and 57 yr, respectively (range, 28-77 yr). They were all diagnosed clinically by biochemical parameters and liver technetium scan. In 42 patients liver needle biopsy was performed. Markers of hepatitis B infection, such as hepatitis B surface antigen, antibody to hepatitis B surface antigen, and antibody to hepatitis B core antigen, were
RIA GH was measured by a RIA kit (Coup. Dris Industrie S.A., Gif-sur-Yvette, France). Insulin-like growth factor-I (IGF-I) was measured by RIA on acid-ethanol-extracted serum, using a nonequilibrium technique (8). The standard was acid-ethanol
Received November 27,1990. Address all correspondence and requests for reprints to: Dr. Yaacov Baruch, Department of Medicine B, Rambam Medical Center, P.O.B. 9602, 31096 Haifa, Israel. 777
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 18:37 For personal use only. No other uses without permission. . All rights reserved.
778
BARUCH ET AL.
extract of pooled normal human sera, used over a range of 100 /xL. This extract was assigned a value of 1 U IGF-I/mL. IGF-I antibody was a gift from P. Gluckman (Auckland, New Zealand). The assay sensitivity was 5 pg, and the intra- and interassay coefficients of variability were less than 2.5% and 3.7%, respectively.
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GH-BP assay GH-BP was measured by a binding assay with dextrancoated charcoal separation, as previously described (5). Briefly, [125I]human GH (hGH; 1 ng) was incubated with human serum (50 nL) in the absence (total binding) or presence (nonspecific binding) of excess unlabeled hGH (1 ng) in a final volume of 270 fih. Incubations were carried out at 4 C for 24 h and were terminated by the addition of 1.0 mL cold dextran-coated charcoal (2% Norit-A charcoal-0.2% Dextran T-70) in 0.01 mol/L phosphate buffer, pH 7.6, on ice for 15 min, followed by centrifugation at 3000 X g for 20 min at 4 C. The supernatant was collected and counted in an automatic 7-counter. The specific binding of [125I]hGH obtained with 50 nL serum was expressed as a percentage of the total counts per min. The sensitivity of the assay was l%/0.02 mL serum, and intra- and interassay variabilities were 1.5% and 4.6%, respectively. In 18 patients serum GH levels were greater than 2 ngfL (5.5 ± 1.2 Mg/L). Their GH-BP results were corrected for serum GH, as previously described (5, 9, 10). Assessment of binding affinity (Ka) and capacity (Bmax) was performed by Scatchard analysis. Statistics Results are expressed as the mean ± SEM. Two-tailed Student's t test was used for intergroup comparisons. Regression and linear correlation coefficients were calculated by the least squares method. P < 0.05 was considered significant.
JCE & M • 1991 Vol 73 • No 4
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PUGH'S SCORE FIG. 1. Correlation of Pugh's score with serum GH-BP levels in cirrhotic patients. • , Patients with both cirrhosis and diabetes mellitus (r = -0.48; P < 0.001); O, patients with cirrhosis only (r = -0.5- P < 0.001). 0
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Results Using Pugh's score, 31 of the 52 cirrhotic patients were classified as class A, with scores of 5 or 6, and 21 patients as class B or C, with scores ranging from 7-15. The mean serum globulin level was 33.6 ± 1 . 2 g/L; alkaline phosphatase was 167 ± 19 IU (normal,
Vol. 73, No. 4 Printed in U.S.A.
Decreased Serum Growth Hormone-Binding Protein in Patients with Liver Cirrhosis YAACOV BARUCH, TAMAR AMIT, PNINA HERTZ, RAFAEL ENAT, MOUSSA B. H. YOUDIM, AND ZEEV HOCHBERG Department of Medicine B, Rambam Medical Center (Y.B., R.E.), and the Rappaport Family Institute for Research in the Medical Sciences and Departments of Medicine (Y.B., R.E.) and Pharmacology (T.A., P.H., M.B.H. Y., Z.H.), Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096 Haifa, Israel
ABSTRACT. The recently characterized GH-binding protein (GH-BP) has an amino acid sequence identical to the extracellular domain of the GH receptor. Serum GH-BP reflects the amount of GH receptors, and the liver seems to be their main source. To evaluate the effect of liver disease on GH-BP, 52 patients with liver cirrhosis were studied. Serum GH-BP was measured by a binding assay with dextran-coated charcoal separation. Levels of GH-BP were correlated against the clinical state, assessed by Pugh's score. The GH-BP of 31 Pugh's class A patients was 9.7 ± 0.5%/50 ML serum, and that of 21 Pugh's class B and C patients was 7.2 ± 0.5%/50 nh serum compared
to 11.3 ± 0.5%/50 jiL serum in age-matched controls. GH-BP correlated negatively with Pugh's score and serum bilirubin, and positively with serum albumin. It did not correlate with serum liver enzymes or serum insulin-like growth factor-I. Scatchard analysis of GH binding to the GH-BP revealed similar binding affinities in Pugh's A, B, and C patients and controls. The binding capacity in cirrhosis was significantly lower than that in controls. We conclude that serum GH-BP is controlled mainly by the liver and can provide an additional measure of disease severity in liver cirrhosis. (J Clin Endocrinol Metab 73: 777780,1991)
A
found in 27 of these patients. Based on both clinical and biochemical parameters, they were classified by Pugh's score (6). Pugh's score is composed of five variables. Two are assessed clinically (severity of ascites and encephalopathy), and three are based on laboratory tests (serum albumin and bilirubin levels and prothrombin time). Each variable is given a value of 1, 2, or 3 for increasing abnormality, and the values of the five variables are summed for each patient. Thus, the best possible score is 5, and the worst is 15. Using a continuous scale from 5-15, we get a finely graded measurement of liver disease severity. For simplicity, it can be presented as three classes: class A, 5-6; class B, 7-10; and class C, 11-15. This score has recently been validated as a predictor of prognosis in liver cirrhosis that is correlated with normal functional liver mass (7). Twelve age-matched healthy volunteers (8 males and 4 females) with normal serum liver enzyme tests, protein levels, and prothrombin time served as controls. All patients and volunteers consented to participate in the study. Diabetes mellitus was diagnosed in 12 patients with fasting blood glucose levels above 8.3 mmol/L.
SPECIFIC GH-binding protein (GH-BP) has recently been characterized in human serum (1, 2). A similar protein in rabbit serum has been found to have an amino acid sequence identical to the extracellular domain of the GH receptor (3). The liver may be an important source of GH-BP, and its serum levels have been shown to be reduced in a small number of patients with cirrhosis of the liver (4, 5). The aim of this work was to evaluate in a large group of patients the impact of reduced functional liver mass on GH-BP and to probe the possible role of GH-BP in the evaluation of the severity of liver cirrhosis.
Subjects and Methods Fifty-two patients (27 males and 25 females) with nonalcoholic liver cirrhosis were included in this study. The mean ages of the men and women were 58 and 57 yr, respectively (range, 28-77 yr). They were all diagnosed clinically by biochemical parameters and liver technetium scan. In 42 patients liver needle biopsy was performed. Markers of hepatitis B infection, such as hepatitis B surface antigen, antibody to hepatitis B surface antigen, and antibody to hepatitis B core antigen, were
RIA GH was measured by a RIA kit (Coup. Dris Industrie S.A., Gif-sur-Yvette, France). Insulin-like growth factor-I (IGF-I) was measured by RIA on acid-ethanol-extracted serum, using a nonequilibrium technique (8). The standard was acid-ethanol
Received November 27,1990. Address all correspondence and requests for reprints to: Dr. Yaacov Baruch, Department of Medicine B, Rambam Medical Center, P.O.B. 9602, 31096 Haifa, Israel. 777
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 18:37 For personal use only. No other uses without permission. . All rights reserved.
778
BARUCH ET AL.
extract of pooled normal human sera, used over a range of 100 /xL. This extract was assigned a value of 1 U IGF-I/mL. IGF-I antibody was a gift from P. Gluckman (Auckland, New Zealand). The assay sensitivity was 5 pg, and the intra- and interassay coefficients of variability were less than 2.5% and 3.7%, respectively.
15
oa: a. o
GH-BP assay GH-BP was measured by a binding assay with dextrancoated charcoal separation, as previously described (5). Briefly, [125I]human GH (hGH; 1 ng) was incubated with human serum (50 nL) in the absence (total binding) or presence (nonspecific binding) of excess unlabeled hGH (1 ng) in a final volume of 270 fih. Incubations were carried out at 4 C for 24 h and were terminated by the addition of 1.0 mL cold dextran-coated charcoal (2% Norit-A charcoal-0.2% Dextran T-70) in 0.01 mol/L phosphate buffer, pH 7.6, on ice for 15 min, followed by centrifugation at 3000 X g for 20 min at 4 C. The supernatant was collected and counted in an automatic 7-counter. The specific binding of [125I]hGH obtained with 50 nL serum was expressed as a percentage of the total counts per min. The sensitivity of the assay was l%/0.02 mL serum, and intra- and interassay variabilities were 1.5% and 4.6%, respectively. In 18 patients serum GH levels were greater than 2 ngfL (5.5 ± 1.2 Mg/L). Their GH-BP results were corrected for serum GH, as previously described (5, 9, 10). Assessment of binding affinity (Ka) and capacity (Bmax) was performed by Scatchard analysis. Statistics Results are expressed as the mean ± SEM. Two-tailed Student's t test was used for intergroup comparisons. Regression and linear correlation coefficients were calculated by the least squares method. P < 0.05 was considered significant.
JCE & M • 1991 Vol 73 • No 4
2 Q 2
m I
x o
12 »o
•
•
9
6
•
:
^
"
^
\
0 1
•
1
L
1
7
I
9
1
1
1
11
13
PUGH'S SCORE FIG. 1. Correlation of Pugh's score with serum GH-BP levels in cirrhotic patients. • , Patients with both cirrhosis and diabetes mellitus (r = -0.48; P < 0.001); O, patients with cirrhosis only (r = -0.5- P < 0.001). 0
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20
30
40
50
60
70
80
BILIRUBIN (umol/l)
Results Using Pugh's score, 31 of the 52 cirrhotic patients were classified as class A, with scores of 5 or 6, and 21 patients as class B or C, with scores ranging from 7-15. The mean serum globulin level was 33.6 ± 1 . 2 g/L; alkaline phosphatase was 167 ± 19 IU (normal,
