GASTROENTEROLOGY
1992;102:1666-1692
Depression of Plasma Gelsolin Level During Acute Liver Injury HIROAKI ITO, HIROSHI KAMBE, YOSHIHIRO KIMURA, HIDEJI NAKAMURA, EIJIRO HAYASHI, TADAMITSU KISHIMOTO, SUSUMU KISHIMOTO, and HIDE0 YAMAMOTO Department
of Medicine
III, Osaka University
Medical School, Osaka City, Osaka, Japan
Human plasma contains two actin-binding proteins, plasma gelsolin and vitamin D-binding protein. These proteins are considered to play an important role in the disposition of actin derived from injured tissue. To evaluate this actin-scavenger system, gelsolin concentrations were measured in serial plasma samples obtained from patients with acute liver injury using an enzyme-linked immunosorbent assay. Plasma gelsolin levels in 43 healthy persons were 226 f 52 pg/mL. They were markedly reduced to 80 f 40 pg/mL in 14 patients with an early stage of acute hepatitis and returned to normal levels of 232 + 38 pg/mL as the disease resolved. Moreover, they showed a significant negative correlation with serum aminotransferase and bilirubin levels. In 7 patients with hepatocellular carcinoma, plasma gelsolin levels rapidly decreased from 182 + 42 to 87 f 41 pg/mL after transcatheter arterial embolization therapy. Because plasma gelsolin is not a hepatic protein, the decreased levels are considered to depend exclusively on the extent of actin leakage from the injured liver.
A
ctin is a major intracellular protein of nonmuscle cells as well as muscle cells.‘-3 Tissue injury may therefore cause a liberation of considerable amount of actin into the blood stream.4-‘0 Although nonmuscle cells contain both globular (G-actin) and filamentous (F-actin) forms of the protein, plasma salt concentrations favor polymerization of G-actin into long filaments, which will do harm to the organism by increasing the blood viscosity.” Plasma contains two actin-binding proteins, plasma gelsolin and vitamin D-binding protein (DBP). Animal experiments have revealed that complexes of actin with these proteins are rapidly cleared from the circulation.“-15 Therefore, plasma levels of these actin-binding proteins are expected to decrease following an extensive tissue injury. Moreover, DBP is considered to play a more important role in clearing actin from the circulation because DBPactin complexes are cleared more quickly than gel-
solin-actin complexes.” Actually, decreased serum DBP levels have been shown in patients with various liver diseases.7’gs’6 However, plasma gelsolin levels have not been examined in the corresponding diseases. Hence, using an enzyme-linked immunosorbent assay (ELISA), we measured plasma gelsolin levels during the course of acute hepatitis and examined a correlation between the depression of plasma gelsolin levels and the severity of ongoing liver damage. We also studied patients with hepatocellular carcinoma (HCC) who underwent transcatheter arterial embolization (TAE) therapy. Materials and Methods Subjects Serial plasma samples were obtained from 14 patients with acute viral hepatitis (hepatitis A, 7; B, 5; non-A, non-B, 2) and 7 patients with HCC who received TAE therapy. Plasma was also obtained from 7 cirrhotic patients without HCC. The peak alanine aminotransferase (ALT) levels in acute hepatitis patients ranged from 406 to 3010 U/L (mean + SD, 1498 of:838 U/L), the peak aspartate aminotransferase (AST) levels from 72 to 2019 U/L (864 -t 637 U/L), and the peak bilirubin levels from 24 to 328 pmol/L (1.4 to 19.2 mg/dL) [144 f 108 lmol/L (8.4 + 6.3 mg/dL)]. All the patients with acute hepatitis made steady progress toward complete cure without any complications. Serial plasma samples were collected until ALT levels became less than twice the upper limit of normal. All the HCC patients were assessed as having liver cirrhosis on the basis of biochemical tests, medical imaging, and longstanding histories of chronic liver disease. Their ALT levels ranged from 38 to 139 U/L (mean & SD, 67 + 31 U/L), AST levels from 47 to 153 U/L (83 f 33 U/L), and indocyanine green retention rates at 15 minutes (ICGR,,) from 24% to 52% (37% f 9%). In the cirrhotic patients without HCC, ALT levels ranged from 11 to 48 U/L (28 t 12 U/L), AST levels from 22 to 220 U/L (65 t 65 U/L), and ICGR,, from 25% to 47% (33% f 7%). Plasma samples from 43 healthy subjects were also examined. Blood was collected by venipuncture with a syringe 0 1992 by the American
Gastroenterological
0016-5085/92/$3.00
Association
PLASMA
May 1992
containing 0.1 mL of 3.8% sodium citrate to a total volume of 1.0 mL. After gentle mixing, the blood was centrifuged at 3000 X g for 20 minutes and plasma was separated. Plasma samples were stored frozen at -2O“C until they were assayed for gelsolin levels. Protein
Preparation
Gelsolin was purified from outdated frozen human plasma by methods including ammonium sulfate fractionation, diethylaminoethyl chromatography, and Cibacron Blue F3GA chromatography.17*” After 70% ammonium sulfate precipitation of the purified protein, the resulting pellet was resuspended with phosphate-buffered saline (PBS) and dialyzed against the same buffer. Protein concentration was determined by the method of Bradfordlg using bovine y-globulin as standard. Polyclonal anti-gelsolin antibody was obtained from a rabbit immunized with human plasma gelsolin, and the antibody was shown to be monospecific to the protein as previously described.‘7r’8
ELISA Plasma gelsolin concentration was measured by an indirect competitive ELKA.*’ After purified plasma gelsolin was diluted to a concentration of 10 pg/mL in 0.1 mol/ L sodium carbonate buffer, pH 9.6, 100 pL of the solution was added to each well in a flat-bottomed microtiter plate and incubated overnight at 4°C. The wells were washed with PBS containing 0.05% Tween 20 (TPBS), and the residual absorption sites on the plastic surface were saturated
0
L-1 1.56
” 3.13
6.25
GELSOLIN
” 12.5
25
” 50
100
(IQ/ mL)
Figure 1. Standard curve (0) for ELISA of plasma gelsolin and dilution curve (0) of human plasma. The standard curve was constructed using serial dilutions of purified plasma gelsolin in TPBS containing 1% BSA. The dilution curve was obtained from plasma of a healthy parson diluted 2.5-160-fold in the same buffer. Absorbance in the ordinate is expressed as the percentage of that in the presence of standard plasma gelsolin or test plasma relative to that in the presence of buffer containing no gelsolin. Data are presented in a semilog plot. Two curves parallel each other at gelsolin concentrations ranging from 3.13 to 50 w/mL.
GELSOLIN
LEVEL
DURING
ACUTE
LIVER
INJURY
1687
by treatment with 1% bovine serum albumin (BSA) in PBS overnight at 4°C. The plate was then washed with TPBS and incubated overnight at 4°C with 50 pL of diluted antigelsolin antibody and an equal volume of standard gelsolin solutions or plasma samples diluted in TPBS containing 1% BSA. Sodium citrate was added to the standard gelsolin solutions to adjust the anticoagulant concentration to that in the plasma samples. After the plate was washed with TPBS, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G (Bio-Rad Labs., Richmond, CA) diluted in TPBS containing 1% BSA was added and incubated for 2 hours at room temperature. The plate was then washed with TPBS, and each well was filled with 100 pL of the enzyme substrate, 2,2-azino-di(3_ethylbenzthiazoline)-sulfonic acid (Zymed Lab., San Francisco, CA) dissolved in 0.1 mol/L citrate buffer, pH 4.2, containing 0.03% hydrogen peroxide. The reaction was developed for 45 minutes and stopped by adding 2 mmol/L sodium azide. The optical density of the fluid was measured at 405 nm in an ImmunoReader NJ2000 spectrophotometer (InterMed Japan, Tokyo). The gelsolin concentration of each sample was calculated by comparing the optical density with a standard curve made by serial dilutions of the purified gelsolin solution. The samples were assayed in triplicate. Detection of Actin-Gelsolin Complexes in Plasma
and Actin-DBP
Detection of actin and its complexes with gelsolin and DBP was performed by the affinity chromatographic method.4,21,22This method is based on the fact that deoxyribonuclease I (DNase I) has a high affinity for G-actin and forms ternary complexes comprising DNase I-actin-gelsolin and DNase I-actin-DBP.Z3.24 A mixture of 100 pL of plasma and 200 yL of 25% (vol/vol) DNase I-Sepharose suspension in 10 mmol/L Tris-buffered saline, pH 7.4, was incubated for 1 hour at 4’C. After washing with 10 mmol/ L Tris-buffered saline, pH 7.4, containing 0.05% Tween 20, the Sepharose was mixed with 60 pL of Laemmli’s gel sample buffer,25 boiled, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins separated on the gel were transblotted to a nitrocellulose sheet according to the method of Towbin et aLz6 and incubated with 5% BSA. Then each strip of sheet was overlaid with rabbit antibody to actin (Transformation Research, Inc., Framingham, MA), gelsolin, and DBP (Dakopatts, Glostrup, Denmark), respectively. The antibodies were detected with the horseradish peroxidase-conjugated goat antibody to rabbit IgG, and the color reaction was performed in PBS containing 0.1% 3,3’-diaminobenzidine tetrahydrochloride and 0.025% hydrogen peroxide. Statistical unpaired
Statistical t tests.
Analysis analysis
was performed
using paired or
Results
Standard
Curve
for ELISA of Plasma
Gelsolin
Figure 1 shows a typical standard curve for ELISA of plasma gelsolin. The curve was constructed
1688 IT0 ET AL.
GASTROENTEROLOGY Vol. 102, No. 5
Table 1. Plasma Gelsolin Levels in Healthy Subjects and Patients With Liver Diseases Diagnosis Healthy Early stage Late stage
226 f 52
14 14
80+40 232 + 38
1
1992;102:1666-1692
Depression of Plasma Gelsolin Level During Acute Liver Injury HIROAKI ITO, HIROSHI KAMBE, YOSHIHIRO KIMURA, HIDEJI NAKAMURA, EIJIRO HAYASHI, TADAMITSU KISHIMOTO, SUSUMU KISHIMOTO, and HIDE0 YAMAMOTO Department
of Medicine
III, Osaka University
Medical School, Osaka City, Osaka, Japan
Human plasma contains two actin-binding proteins, plasma gelsolin and vitamin D-binding protein. These proteins are considered to play an important role in the disposition of actin derived from injured tissue. To evaluate this actin-scavenger system, gelsolin concentrations were measured in serial plasma samples obtained from patients with acute liver injury using an enzyme-linked immunosorbent assay. Plasma gelsolin levels in 43 healthy persons were 226 f 52 pg/mL. They were markedly reduced to 80 f 40 pg/mL in 14 patients with an early stage of acute hepatitis and returned to normal levels of 232 + 38 pg/mL as the disease resolved. Moreover, they showed a significant negative correlation with serum aminotransferase and bilirubin levels. In 7 patients with hepatocellular carcinoma, plasma gelsolin levels rapidly decreased from 182 + 42 to 87 f 41 pg/mL after transcatheter arterial embolization therapy. Because plasma gelsolin is not a hepatic protein, the decreased levels are considered to depend exclusively on the extent of actin leakage from the injured liver.
A
ctin is a major intracellular protein of nonmuscle cells as well as muscle cells.‘-3 Tissue injury may therefore cause a liberation of considerable amount of actin into the blood stream.4-‘0 Although nonmuscle cells contain both globular (G-actin) and filamentous (F-actin) forms of the protein, plasma salt concentrations favor polymerization of G-actin into long filaments, which will do harm to the organism by increasing the blood viscosity.” Plasma contains two actin-binding proteins, plasma gelsolin and vitamin D-binding protein (DBP). Animal experiments have revealed that complexes of actin with these proteins are rapidly cleared from the circulation.“-15 Therefore, plasma levels of these actin-binding proteins are expected to decrease following an extensive tissue injury. Moreover, DBP is considered to play a more important role in clearing actin from the circulation because DBPactin complexes are cleared more quickly than gel-
solin-actin complexes.” Actually, decreased serum DBP levels have been shown in patients with various liver diseases.7’gs’6 However, plasma gelsolin levels have not been examined in the corresponding diseases. Hence, using an enzyme-linked immunosorbent assay (ELISA), we measured plasma gelsolin levels during the course of acute hepatitis and examined a correlation between the depression of plasma gelsolin levels and the severity of ongoing liver damage. We also studied patients with hepatocellular carcinoma (HCC) who underwent transcatheter arterial embolization (TAE) therapy. Materials and Methods Subjects Serial plasma samples were obtained from 14 patients with acute viral hepatitis (hepatitis A, 7; B, 5; non-A, non-B, 2) and 7 patients with HCC who received TAE therapy. Plasma was also obtained from 7 cirrhotic patients without HCC. The peak alanine aminotransferase (ALT) levels in acute hepatitis patients ranged from 406 to 3010 U/L (mean + SD, 1498 of:838 U/L), the peak aspartate aminotransferase (AST) levels from 72 to 2019 U/L (864 -t 637 U/L), and the peak bilirubin levels from 24 to 328 pmol/L (1.4 to 19.2 mg/dL) [144 f 108 lmol/L (8.4 + 6.3 mg/dL)]. All the patients with acute hepatitis made steady progress toward complete cure without any complications. Serial plasma samples were collected until ALT levels became less than twice the upper limit of normal. All the HCC patients were assessed as having liver cirrhosis on the basis of biochemical tests, medical imaging, and longstanding histories of chronic liver disease. Their ALT levels ranged from 38 to 139 U/L (mean & SD, 67 + 31 U/L), AST levels from 47 to 153 U/L (83 f 33 U/L), and indocyanine green retention rates at 15 minutes (ICGR,,) from 24% to 52% (37% f 9%). In the cirrhotic patients without HCC, ALT levels ranged from 11 to 48 U/L (28 t 12 U/L), AST levels from 22 to 220 U/L (65 t 65 U/L), and ICGR,, from 25% to 47% (33% f 7%). Plasma samples from 43 healthy subjects were also examined. Blood was collected by venipuncture with a syringe 0 1992 by the American
Gastroenterological
0016-5085/92/$3.00
Association
PLASMA
May 1992
containing 0.1 mL of 3.8% sodium citrate to a total volume of 1.0 mL. After gentle mixing, the blood was centrifuged at 3000 X g for 20 minutes and plasma was separated. Plasma samples were stored frozen at -2O“C until they were assayed for gelsolin levels. Protein
Preparation
Gelsolin was purified from outdated frozen human plasma by methods including ammonium sulfate fractionation, diethylaminoethyl chromatography, and Cibacron Blue F3GA chromatography.17*” After 70% ammonium sulfate precipitation of the purified protein, the resulting pellet was resuspended with phosphate-buffered saline (PBS) and dialyzed against the same buffer. Protein concentration was determined by the method of Bradfordlg using bovine y-globulin as standard. Polyclonal anti-gelsolin antibody was obtained from a rabbit immunized with human plasma gelsolin, and the antibody was shown to be monospecific to the protein as previously described.‘7r’8
ELISA Plasma gelsolin concentration was measured by an indirect competitive ELKA.*’ After purified plasma gelsolin was diluted to a concentration of 10 pg/mL in 0.1 mol/ L sodium carbonate buffer, pH 9.6, 100 pL of the solution was added to each well in a flat-bottomed microtiter plate and incubated overnight at 4°C. The wells were washed with PBS containing 0.05% Tween 20 (TPBS), and the residual absorption sites on the plastic surface were saturated
0
L-1 1.56
” 3.13
6.25
GELSOLIN
” 12.5
25
” 50
100
(IQ/ mL)
Figure 1. Standard curve (0) for ELISA of plasma gelsolin and dilution curve (0) of human plasma. The standard curve was constructed using serial dilutions of purified plasma gelsolin in TPBS containing 1% BSA. The dilution curve was obtained from plasma of a healthy parson diluted 2.5-160-fold in the same buffer. Absorbance in the ordinate is expressed as the percentage of that in the presence of standard plasma gelsolin or test plasma relative to that in the presence of buffer containing no gelsolin. Data are presented in a semilog plot. Two curves parallel each other at gelsolin concentrations ranging from 3.13 to 50 w/mL.
GELSOLIN
LEVEL
DURING
ACUTE
LIVER
INJURY
1687
by treatment with 1% bovine serum albumin (BSA) in PBS overnight at 4°C. The plate was then washed with TPBS and incubated overnight at 4°C with 50 pL of diluted antigelsolin antibody and an equal volume of standard gelsolin solutions or plasma samples diluted in TPBS containing 1% BSA. Sodium citrate was added to the standard gelsolin solutions to adjust the anticoagulant concentration to that in the plasma samples. After the plate was washed with TPBS, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig) G (Bio-Rad Labs., Richmond, CA) diluted in TPBS containing 1% BSA was added and incubated for 2 hours at room temperature. The plate was then washed with TPBS, and each well was filled with 100 pL of the enzyme substrate, 2,2-azino-di(3_ethylbenzthiazoline)-sulfonic acid (Zymed Lab., San Francisco, CA) dissolved in 0.1 mol/L citrate buffer, pH 4.2, containing 0.03% hydrogen peroxide. The reaction was developed for 45 minutes and stopped by adding 2 mmol/L sodium azide. The optical density of the fluid was measured at 405 nm in an ImmunoReader NJ2000 spectrophotometer (InterMed Japan, Tokyo). The gelsolin concentration of each sample was calculated by comparing the optical density with a standard curve made by serial dilutions of the purified gelsolin solution. The samples were assayed in triplicate. Detection of Actin-Gelsolin Complexes in Plasma
and Actin-DBP
Detection of actin and its complexes with gelsolin and DBP was performed by the affinity chromatographic method.4,21,22This method is based on the fact that deoxyribonuclease I (DNase I) has a high affinity for G-actin and forms ternary complexes comprising DNase I-actin-gelsolin and DNase I-actin-DBP.Z3.24 A mixture of 100 pL of plasma and 200 yL of 25% (vol/vol) DNase I-Sepharose suspension in 10 mmol/L Tris-buffered saline, pH 7.4, was incubated for 1 hour at 4’C. After washing with 10 mmol/ L Tris-buffered saline, pH 7.4, containing 0.05% Tween 20, the Sepharose was mixed with 60 pL of Laemmli’s gel sample buffer,25 boiled, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins separated on the gel were transblotted to a nitrocellulose sheet according to the method of Towbin et aLz6 and incubated with 5% BSA. Then each strip of sheet was overlaid with rabbit antibody to actin (Transformation Research, Inc., Framingham, MA), gelsolin, and DBP (Dakopatts, Glostrup, Denmark), respectively. The antibodies were detected with the horseradish peroxidase-conjugated goat antibody to rabbit IgG, and the color reaction was performed in PBS containing 0.1% 3,3’-diaminobenzidine tetrahydrochloride and 0.025% hydrogen peroxide. Statistical unpaired
Statistical t tests.
Analysis analysis
was performed
using paired or
Results
Standard
Curve
for ELISA of Plasma
Gelsolin
Figure 1 shows a typical standard curve for ELISA of plasma gelsolin. The curve was constructed
1688 IT0 ET AL.
GASTROENTEROLOGY Vol. 102, No. 5
Table 1. Plasma Gelsolin Levels in Healthy Subjects and Patients With Liver Diseases Diagnosis Healthy Early stage Late stage
226 f 52
14 14
80+40 232 + 38
1
