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Clinica Chimicu Acra, 210 (1992) 109-118 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/92/%05.00 CCA 05363
Concentration of serum laminin and type IV collagen in liver diseases assayed by a sandwich enzyme-immunoassay using monoclonal antibodies Yukihiro Yokoyaa, Kazushi Iwatac, Yasuteru Muragakia, Chiyo Shiota”, Yoshifumi Morimoto”, Masaya Inou.ea, Hidekazu Itohb, Shingo Nishiokab and Akira Ooshima” ‘Department
of Pathology, ‘The second Division of Internal Medicine, Wakuyama Medical Wakayama and ‘Fuji Chemical Industries, Ltd., Takaoka, Toyama (Japan]
College,
(Received 15 February 1992; revision received 3 June 1992; accepted 20 June 1992)
Key words: Sandwich enzyme-immunoassay;
Human laminin; Human type IV collagen; Liver disease; Monoclonal antibody
Summary Serum laminin (Pl fragment) and type IV collagen levels were determined in patients with hepatic disorders. The method was based on a sandwich enzymei~unoassay using two monoclonal antibodies that recognize different epitopes of either laminin or type IV collagen molecule. Laminin and type IV coliagen levels in the serum of patients with chronic hepatic disorders were higher as compared with those in healthy control subjects, with the increment of serum type IV collagen being far greater than that of laminin. Since type IV collagen and laminin are major basement membrane components, it is suggested that the higher levels of these peptides may reflect a so-called capillarization of the ~risinusoidal wall encountered in hepatic Iibrogenesis. The assay system used in this experiment is simple and sensitive and can be applied to clinical evaluation of hepatic fibrosis.
Increased deposition of extracellular matrices including various types of collagen and laminin is the main feature of hepatic fibrosis. Collagen and laminin biosynCorrespondence to: Dr. Y. Yokoya, Department of Pathology, Wakayama Medical College, 9-Bancho, Wakayama 640, Japan.
110
thesis is elevated both in human liver disorders and experimentally induced liver injury [l-7]. Competitive radioimmunoassay with polyclonal antibodies have been used to demonstrate increased levels of serum laminin (Pl fragment) and type IV collagen in human hepatic disorders [8-141. In this experiment, we have adopted a sandwich enzyme-immunoassay with monoclonal antibodies recently developed [15- 181. The serum levels of laminin and type IV collagen were closely correlated and increased in patients with chronic hepatic disorders as compared with normal control subjects. Comparisons were made for each of the hepatic disorders listed under patients. We also attempted to determine whether these parameters could be used as serological markers of hepatic fibrosis. Patients and assays Patients
A sandwich enzyme-immunoassay of serum laminin and type IV collagen was performed in 96 control subjects (CS), 20 patients with acute hepatitis (AH), 5 with fulminant hepatitis (FH), 12 with chronic inactive hepatitis (CIH), 45 with chronic active hepatitis (CAH), 43 with liver cirrhosis (LC) and 40 with liver cirrhosis
I-
-
Laminin
-
Type
IV
col
I agen MfsD
203
(5) 1
(16)
lo-
20-
(9) 30-
(10)
03)
40-
Age
50-
in
(10)
I
60-
(2) 70-
(11) 80-
years
Fig. I. Age-related changes in serum laminin and type IV collagen (control subjects). Statistically significant as compared to the mean value of age range from 20 to 69 years old. *,**P < 0.05, P < 0.01, respectively. ( ): Number of subjects examined.
III
associated with hepatocellular carcinoma (LC + HCC). Classification was based on clinical, biochemical, histological, ultrasound and computed tomographical findings. CIH and CAH were confirmed by histological examination. Control sera were obtained from healthy subjects without hepatic, renal or endocrine dysfunction. In the control subjects, an age-related change of serum laminin and type IV collagen was noted. Assays
Serum was stored at -80°C until analysed. Serum laminin and type IV collagen was measured by the sandwich enzyme-imm?noassays recently developed [ 1% 181. In brief, the assay system used monoclonal antibodies as a solid phase (clone 22-
. . .
.
. .
.
--kA
. . ---.
ItT 1 (58)
(20)
(5)
CS
AH
FH
8
--_---e
-tc -T
--f
t=:
3.
(12)
(45)
(43)
CIH
CAH
LC
(40 1 LC+HCC
Fig. 2. Serum laminin in patients with hepatic disorders and control subjects. Horizontal bar indicates the mean value of each group. Broken line indicates the upper limit of M + 2 SD. in control subjects. ( ): Number of cases examined. CS: control subjects, AH: acute hepatitis, FH: fulminant hepatitis, CIH: chronic inactive hepatitis, CAH: chronic active hepatitis, LC: liver cirrhosis, LC + HCC: liver cirrhosis associated with hepatocellular carcinoma.
112
3Bll; anti-laminin, clone 4H12; anti-type IV collagen). Wells of the microplates (Micro-Well Module in frame, Nunc, Denmark) were coated with 100 ~1 purified monoclonal antibody (10 pg/ml) in 20 mM carbonate buffer (pH 9.6) containing 0.02% sodium azide at 4°C for 24 h and washed with PBS containing 0.05% Tween 20. Then the microplates were blocked with 1% BSA in PBS and washed and incubated with 20 ~1 solution containing either serum samples or standard antigens and with 100 ~1 peroxidase-conjugated Fab’ of mouse monoclonal antibody (clone HL-4H3; anti-laminin, clone lD3; anti-type IV collagen) in PBS (1 &ml) for 1 h at room temperature. After washing, 100 ~1 substrate buffer [0.02% (v/v) H202, 0.1 mg/ml o-phenylenediamine and 1% (v/v) methanol in 0.05 M Na-citrate buffer, pH 5.01 was added to the wells and incubated for 15 min. The reaction was stopped with
. c .
.
.a
.
.
. . .
.
. . ..
(5) AH
I. . .. .
T f
:
CS
t
FH
(12) CIH
(45)
(43)
CAH
LC
.. -. ; l
ma ..
. F
7
(40) LC+HCC
Fig. 3. Serum type IV collagen in patients with hepatic disorders and control subjects. Horizontal bar indicates the mean value of each group. Broken line indicates the upper limit of M + 2 S.D. in control subjects. ( ): Number of cases examined. CS: control subjects, AH: acute hepatitis, FH: fulminant hepatitis, CIH: chronic inactive hepatitis, CAH: chronic active hepatitis, LC: liver cirrhosis, LC + HCC: liver cirrhosis associated with hepatocellular carcinoma.
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50 ~1 of 4 N H2S04. The absorbance at 492 nm was measured by a microplate reader. The concentrations of laminin and type IV collagen were calculated from the corresponding standard curve. All tests were carried out in duplicate. Statistical analysis Comparison of assay data among groups was performed using Student’s r-test. Correlations were determined by a linear regression analysis. Results In the control subjects, serum laminin and type IV collagen were slightly elevated in the subjects aged from 10 to 19 years and from 70 to 89 years compared with those in the subjects aged from 20 to 69 years (Fig. l), where serum laminin concentrations ranged from 84 to 180 ng/ml (mean 131.7 @ml). There were no significant differences between male and female in the level of these markers. In the sera from patients with AH, FH, CIH, CAH, LC and LC + HCC, the concentrations were 147.7 f 50.4 @ml, 355.2 f 218.6 @ml, 136.7 f 33.4 @ml, 211.0 + 90.3 q/ml, 248.1 f 90.3 q/ml and 265.5 f 103.1 q/ml, respectively (Fig. 2), with the higher -
1
I &-o *.-+
KC with LC HCC without LC Partial hepatectomy (HCC
800-
with
LC)
r S
8._ g al 0.
600-
B ._ W 8 P .E ._ I A
I
t!
4.
S P
3 \
\
600-
\
5
400-
-0
; 400-
L $ r-” 200 -
200 -
*_--
_____--I
0
I
12 Interval of (months)
I
24 assays
1
I
_a
-‘-.___.; __--
I
I
0
12 Interval of (months)
I
24 assays
Fig. 4. Variation of serum laminin and type IV collagen during clinical course of hepatocellular carcinoma (HCC) with or without liver cirrhosis (LC).
*
Laminin
peptide
cs
CGzml)
n=56 r=O.29 PXO.05 y=o.27xt4a.4 1
Laminin
peptide
AH
(ng/ml)
xl0
I
/
Laminin
I
(ng/ml)
3w
-1,.
n=12 r=O.77 P ranges CAH:
;:{:;:$$:a:::::Y=1.23Xt86.3 ::::::::::::::::::; I 1 500
:::+:;:::::::>..; P~:~,~~~~~~ .
.
.
LCtHCC
Fig. 5. Relationship between serum laminin and type IV collagen in patients with hepatic disorders and control subjects. Shaded area includes normal of serum laminin and type IV collagen (M + 2 SD.). CS: control subjects, AH: acute hepatitis, FH: fulminant hepatitis, CIH: chronic inactive hepatitis, chronic active hepatitis, LC: liver cirrhosis, LC + HCC: liver cirrhosis associated with hepatocellular carcinoma.
Laminin
CAH
116
levels in patients with FH (P < 0.05) CAH (P < 0.001) LC (P < 0.001) and LC + HCC (P < 0.001). In particular, the levels were significantly higher in CAH than in CIH (P < 0.001) and in LC than in CAH (P < 0.05). No significant difference was found between laminin levels in LC with or without HCC. Serum type IV collagen ranged from 40 to 128 @ml (mean 84.2 @ml) in the subjects aged from 20 to 69 years. Type IV collagen in the sera from patients with AH, FH, CIH, CAH, LC and LC + HCC were 126.3 f 64.3 ng/ml, 318.0 f 106.8 @ml, 136.6 f 60.8 @ml, 217.1 f 135.0 q/ml, 260.6 f 145.1 @ml and 411.7 f 238.7 rig/ml, respectively (Fig. 3), with significantly higher concentrations in AH (P < 0.01) FH (P < O.OOl),CIH (P < 0.01) CAH (P < O.OOl), LC (P < 0.001) and LC + HCC (P c 0.001). In CAH, the levels were significantly higher than in CIH (P < 0.01) and statistically higher value (P < 0.001) was noted in LC + HCC than in LC alone. In hepatocellular carcinoma with liver cirrhosis, the serum levels of laminin and type IV collagen both tended to increase gradually and in a patient who had partial hepatectomy, both markers were somewhat lower (Fig. 4). Serum laminin and type IV collagen were closely correlated. Using these two markers, the populations that exceed the normal value (M + 2 S.D.) were 40% in AH, 100% in FH, 36.7% in CIH, 86.4% in CAH, 86% in LC and 100% in LC + HCC (Fig. 5). When the ratio of type IV collagen to laminin was calculated, the value tended to increase with the progress of fibrosis: the percentage of cases exceeding the ratio of 2 in patients with CAH, LC and LC + HCC were 4.5%, 2.3% and 30.0%, respectively (Table I). Additionally, both markers showed slightly positive correlation with such biochemial liver parameters as y-GTP, T.Bil and ICGRis and negative correlation with CES (data are not shown).
TABLE Ratio
I of type IV collagen
to laminin
in patients
with hepatic
FH (5) CIH (12) CAH (45) LC (43) LC + HCC (40)
0.66 0.87 1.18 0.99 1.04 1.07 1.63
* zt zt f f f f
0.19 0.32** 0.59 0.28*** 0.44*** 0.45*** 0.87”;
and control
subjects.
Percent of cases exceeding ratio of
Type IV collagen/ Laminin
CS (58) AH (20)
disorders
%§ §% I
11 $c&
1-l
5
I.0
2.0
5.2% 25.0 60.0 41.7 43.2 51.2 75.0
0% 0 0 0 4.5 2.3 30.0
the
Statistically significant as compared to control value. *** ***P< 0.01, P < 0.001, respectively. #*HP < 0.05, P < 0.001, respectively. ( ): Number of cases examined. M * SD. CS: control subjects, AH acute hepatitis, FH: fulminant hepatitis, CIH: chronic inactive hepatitis, CAH: chronic active hepatitis, LC: liver cirrhosis, LC + HCC: liver cirrhosis associated with hepatocellular carcinoma.
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Discussion Chronic liver disease is characterized by repeated hepatocellular injury, inflammation and fibrosis [l-2]. Increased deposition of basement membrane components such as type IV collagen and laminin in the perisinusoidal wall of the liver may cause hepatocellular dysfunction and portal hypertension [19-211. It is, therefore, important to grasp the dynamics of basement membrane metabolism in making the original diagnosis, as well as in determining the efficacy of treatment and evaluating prognosis. In this study, serum concentrations of laminin and type IV collagen were measured in 165 patients with various liver diseases and in control subjects. Serum levels of laminin and type IV collagen were both significantly higher in patients with FH, CAH, LC, or LC + HCC than in the healthy controls, the concentrations of serum laminin and type IV collagen increased in relation to the progression of hepatic fibrosis. The levels were significantly higher in patients with CAH than in those with CIH and in those.with LC associated HCC the serum level of laminin and type IV collagen were markedly elevated. Broks et al. [22] have also reported that serum laminin increases in about 50% of cancer patients. This may be explained by the destruction of the basement membrane by tumor invasion and increased synthesis of the fibrous component surrounding the tumor. Matuda [23] has shown that serial measurement of laminin in hepatocellular carcinoma is useful for evaluating the effectiveness of transarterial embolization (TAE). In our patients with FH, the serum levels of both markers were significantly elevated. This may reflect the destruction of the basement membrane due to multiorgan failure and increased synthesis of the basement membrane accompanying the regeneration of hepatocytes. It has been demonstrated in CCl&reated rats that serum type IV collagen and laminin rapidly increase after hepatocellular damage [6,7,16]. The change of type IV collagen was greater than that of laminin after CC&-treatment, suggesting that each peptide yields a different synthetic or turnover rate. For practical use, we have calculated an arbitrary value of type IV collagen/laminin ratio; ratios exceeding 2.0 seem to be a useful parameter of hepatic fibrosis. Finally, the assay system employed in this experiment was simple and sensitive and can be applied to monitoring hepatic tibrogenesis. References 1 2 3 4 5 6
Popper H, Udenfriend S. Hepatic fibrosis: Correlation of biochemical and morphologic investigations. Am J Med 1970;49:707-721. Popper N. Pathologic aspects of cirrhosis: A review. Am J Path01 1977;87:228-264. Rojkind M, Martinez-Palomo A. Increase in type I and type III collagens in human alcoholic liver cirrhosis. Proc Nat1 Acad Sci USA 1976;73:539-543. Kent G, Gay S, Inouye T et al. Types 1 and type III collagen in experimental liver injury. Gastroenterology 1976;71:915. Hahn E, Wick G, Pencev D, Timpl R. Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin and tibronectin. Gut 1980;21:63-71. Martinez-Hernandez A. The hepatic extracellular matrix. II. Electron immunohistochemical studies in rats with Ccl,-induced cirrhosis. Lab Invest 1985;53:166-186.
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12
13
14
I5 16
17 18
19 20 21 22 23
Matsumoto E, Muragaki Y, Ooshima A. Increased serum type IV collagen peptide in carbon tetrachloride-treated rats. Acta PathoI Jpn 1989:39:23-29. Niemell 0, Risteli L, Sotaniemi EA, Risteli J. Type IV collagen and laminin-related antigens in human serum in alcoholic liver disease. Em J Clin Invest 1985;15:132-137. Gressner AM and Tittor W. Serum laminin - its concentration increases with portal hypertension in cirrhotic hver disease. Klin Wochenschr 198664: 1240- 1248. Van Zanten RAA. van Leeuwen RE, Wilson JHP. Serum procoltagen III N-termina1 peptide and laminin Pl fragment concentrations in alcoholic liver disease and primary biiiary cirrhosis, Clin Chim Acta 1988;177:141-146. Schneider M. Voss B. Hogemann B, Eberhardt G, Gerlach U. Evaluation of serum laminin PI, procollagenpeptides and ~-acetyl-~-glucosaminidase for monito~ng the activity of liver fibrosis. Hepatogastroenterol 1989;36:506-510. Niemela 0, Risteli J. Blake JE. Risteli L. Compton KV. Orrego H. Markers of fibrogenesis and basement membrane formation in alcoholic liver disease - Relation to severity, presence of hepatitis and alcohol intake. Gastroenterology 1990,98:1612-1619. Nakano K, Nakagawa M, Nakabayashi H, Okamoto T, Takamatsu S, Fukuda Y. Serum levels of laminin PI in normal subjects and patients with hepatic fibrosis - A reliable and non-invasive marker of liver cirrhosis. Acta Hepatol Jpn 1987;28:1047-1050 (in Japanese). Maruyama K, Okazaki I, Kamegaya K et al. Serum levels of laminin as a tool of detection for ir~versibjlity of hepatic fibrosis in patients with alcoholic liver disease. Acta Hepatol Jpn 1987;28:1.544-1549 (in Japanese}. Obata K, Iwata K, Ichida T et al. One step sandwich enzyme immuno assay for human type IV collagen using monoclonal antibodies. Clin Chim Acta 1989;181:293-304, Matsumoto E, Muragaki Y, Ooshima A. Increased amount of serum type IV collagen peptide in human liver fibrosis as determined by enzyme - immuno assay with mon~lonal antibodies. Acta Path01 Jpn 1989;39:217-223. Fujimoto N, Sejima H, Kotani M, Iwata K, Ichida T, Ooshima A. One-step sandwich enzyme i~unoassay for human laminin using monoclonal antibodies. Clin Chim Acta 1990;191:21I-220. Yokoya Y. Laminin and liver fibrosis - Immunohistologicai study and development of immunoassay system with monoclonal antibodies. J Wakayama Med Sot 1991;42:357-368 (in Japanese). Schaffner F, Popper H. Capillarization of hepatic sinusoids in man. Gastroenterology i963;44:239-242. Phillips MJ, Steiner JW. Electron microscopy of cirrhotic nodule. TubuIarization of the parencbyma by biliary hepatocytes. Lab Invest 1966;15:801-817. Bianchi FB, Biagini G, Ballardini Get al. Basement membrane production by hepatocytes in chronic liver disease. Hepatology 19844: 1167-I 172. Brocks IX, Strecker H, Neubauer HP, Timple R. Radioi~unoassay of laminin in serum and its application to cancer patients. Clin Chem 1986;32:787-791. Matsuda H. Seriat changes in serum level of laminin and type III procollagen N-terminal peptide after transarterial embolization (TAE) in hepatocellular carcinoma. Jpn J Gastroenterol 1991;88:153-161 (in Japanese).