Journal of Gastroenterology and Hepatology (1992) 1, 596-601
LIVER A N D BILIARY
Clinical significance of the serum levels of the 7s domain of type IV collagen in patients with primary biliary cirrhosis T. F U K U T O M I , * S. S A K A M O T O , * H. I S O B E , t H. SAKAI,* A. M A S U M O T O * A N D H. NAWATA* *Third Department of Internal Medicine, Kyushu University, Fukuoka, Japan and tDepartment of Internal Medicine, Fukuoka Saiseikai Hospital, Fukuoka, Japan
Abstract The serum levels of the 7 s domain of type IV collagen were measured with a radio-immunoassay in 42 patients with primary biliary cirrhosis (asymptomatic: n = 28; symptomatic: n = 14), 10 patients with chronic active hepatitis, 10 patients with liver cirrhosis and 10 healthy female controls. Serum levels of the 7 s domain of type IV collagen were: 4.28 ng/mL (3.88-4.72 ng/mL; mean and range of mean k s.d.1 in healthy controls; 5.97 ng/mL (5.07-7.02 ng/mL) in patients with chronic active hepatitis; 8.23 ng/mL (6.40-10.58 ng/mL) in patients with liver cirrhosis; and 6.79 ng/mL (4.76-9.67 ng/mL) in patients with primary biliary cirrhosis. Patients with liver cirrhosis and primary biliary cirrhosis had higher levels of serum 7 s domain of type IV collagen than healthy controls (P < 0.001, respectively). Serum levels of the 7 s domain of type IV collagen in patients with asymptomatic primary biliary cirrhosis, 5.83 ng/mL (4.55-7.48 ng/mL) were significantly lower than those in symptomatic primary biliary cirrhosis, 9.18 ng/ m L (6.53-12.91 ng/mL; P < 0.001). Serum levels of the 7 s domain of type IV collagen increased significantly along with advancement of the histological stages of primary biliary cirrhosis. Serum levels of the 7 s domain of type IV collagen in the paired sera of eight patients with asymptomatic primary biliary cirrhosis (mean interval 30 months, range 12-48 months) showed significant rises during the intervals ( P < 0.05), while serum levels of albumin and total bilirubin did not change significantly during these intervals. It is concluded that serum levels of the 7 s domain of type IV collagen were good markers of fibrosis and may be useful for evaluation of the clinical status in the patients with primary biliary cirrhosis. Key words: 7 s domain of type IV collagen, primary biliary cirrhosis.
INTRODUCTION Hepatic fibrosis develops as a consequence of chronic inflammation and hepatocyte necrosis in the liver. An excessive extracellular matrix deposition during the development of fibrosis has been demonstrated in several chronic liver diseases.’-4 Type IV collagen is part of the extracellular matrix which forms the core of the basement membranes. In normal human liver, type IV collagen is distributed in the basement membranes of blood vessels, bile ducts and ductules and around nerve axons. It also exists in the perisinusoidal space, but does not exist in hepatocytes or in most of the interstitial matrix of the portal fields.’-4 Electron microscopy shows that sinusoids in normal human liver lack typical basement membranes, although type IV collagen is discontinuously demonstrated by immunohistochemical study.5 The lack of basement membranes in sinusoids is considered to allow intimate contact between the blood
and parenchymal cells. However, in fibrotic liver, type IV collagen with laminin becomes so prominent in the perisinusoidal space that it develops true basement membrane^,^-^ a process known as a capillarization of s i n ~ s o i d sThis . ~ process interferes with the interchange of material between the blood and the hepatocytes and causes hepatic dysfunction. Type IV collagen is organized in a network fashion; four amino terminal ends of type IV collagen are linked together to form a highly protease resistant structure that is known as the 7 s domain. Two carboxyterminal ends of type IV collagen are combined to form Non-Collagenous Domain 1 (NC 1).6 Recently several radio-immunoassays have been developed that detect the 7 s domain and N C 1 in serum.’-* Elevated serum levels of the 7 s domain and N C 1 were reported in patients with chronic liver disease and it has been proposed that serum levels of type IV collagen are a good marker of hepatic fibrosis.’-’* But in patients with primary biliary cirrhosis (PBC) there has been only one report on serum levels of the 7 s domain of
Correspondence: Shigeru Sakamoto, Third Department of Internal Medicine, Kyushu University, Maidashi 3-1-1, Fukuoka 812, Japan. Accepted for publication 13 June 1992.
Serum levels of the 7s domain of type IV collagen type IV c01lagen.l~The clinical significance of serum levels of the 7 s domain of type IV collagen in PBC has not been established. In this study we measured the serum levels of the 7 s domain of type IV collagen with a radio-immunoassay using polyclonal antibody to the 7 s domain of type IV collagen purified from human placenta in 42 patients with PBC. Its clinical implication in PBC was also evaluated.
597 method. Briefly, assay procedures were as follows: 100 pL of rabbit polyclonal anti-7S domain of type IV collagen antibody (30 000-fold dilution) was added to 200 p L of serum and incubated at room temperature for 16 h. Then 100 p L of [‘251]-labelled 7 s domain of type IV collagen was added, and incubated at 37°C for 2 h. Free and bound antigens were separated by goat anti-rabbit IgG with polyethylene glycols as sediment retainers. The radioactivity in the pellet was counted. T h e intra- and inter-assay coefficient variations were 4.2 and 6.5%, respectively.
PATIENTS A N D METHODS Patients
Laboratory tests
Forty-two patients (female: n = 41; male: n = 1; age range: 30-78 years; mean age: 54 years) who fulfilled established clinical, biochemical, immunological and histological criteria for the diagnosis of PBC were chosen for this ~ t u d y . ’ ~ Twenty-two -’~ were inpatients and 20 were outpatients. Patients with jaundice and/or pruritus were classified symptomatic PBC (s-PBC), whereas patients lacking those symptoms were classified asymptomatic PBC (a-PBC). The patients who were complicated with collagen disease (rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus) known to raise serum levels of the 7 s domain of type IV collagen independently, or had been treated with anti-fibrotic agents (glucocorticoids, colchicine), had positive tests for (HBsAg) or anti c-100, were not included in this study. The patients with increased serum levels of creatinine were also excluded. Ten patients with chronic active hepatitis (CAH), 10 patients with liver cirrhosis (LC) and 10 healthy subjects were chosen as controls. They were all female and age-matched. Diagnosis of CAH and LC was done from biochemical and histological findings. Six patients with CAH and three patients with LC were type B, and four patients with CAH and seven patients with LC were type C.
Serum albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), y-glutamyl transpeptidase (y-GTP), total bilirubin (T.Bi1) and cholesterol were measured by autoanalyser, Toshiba biochemical analyser (Toshiba, Tokyo, Japan). Serum IgM levels were analysed by nephelometry and antimitochondrial antibody (AMA) was detected by indirect immunofluorescence.
Blood sampling Sera were obtained from all patients and controls and stored at - 20°C until assayed. In eight patients with a-PBC, sera were taken at initial diagnosis and during follow-up consultation. The mean follow-up intervals of these eight patients was 30 months (range 12-48 months). These patients remained asymptomatic during these intervals.
Type IV collagen assay The serum levels of the 7 s domain of type IV collagen were determined with commercially available RIA kits (Type IV collagen 7 s kit, Nippon DPC Corp., Tokyo, Japan) according to the manufacturer’s instructions. Details of this assay have been ”uescribed in a previous publication.I2 The 7 s domain of type IV collagen used in this assay was isolated from human placenta and purified as described previously.16 T h e 7 s domain of type IV collagen was iodinated according to the lactoperoxidase
Liver histology Percutaneous liver biopsy with a Tru-cut needle was performed in 36 of 42 patients with PBC for diagnosis. Tissue sections were stained with hematoxylin-eosin and examined by pathologists in our hospital. Histological grading was done according to Scheuer’s classification. Stage I1 and I11 were grouped together because it was difficult to separate them.”
Statistical analysis Logarithmic transformation was performed for statistical calculations to achieve a normal distribution of serum levels of the 7 s domain of type IV collagen. The data in the figures and text are presented as mean and range (mean - s.d. to mean + s.d.). One way analysis of variance (ANOVA) was used to analyse the statistical significance of the differences of the mean levels of serum 7 s domain of type IV collagen between four groups (controls, CAH, LC, PBC) or three groups (controls, a-PBC, s-PBC). Significance was then tested by the Bonferroni method. The correlation between the 7 s domain of type IV collagen and histological stages of PBC was analysed using Pearson’s correlation coefficient. T h e correlations between the 7 s domain of type IV collagen and the liver function test (ALB, T.Bil, ALT, AST, ALP, and y-GTP) were analysed using Pearson’s correlation coefficient. T o achieve a normal distribution, logarithmic transformation was performed on serum T.Bi1, ALT, AST, ALP, and y-GTP before the statistical calculation. The data on serum ALB were not transformed because they were already normal in distribution. T h e significance of the change of the serum levels of the 7 s domain of type IV collagen during follow-up was anal ysed using the Wilcoxon signed-rank test. All tests were considered statistically significant at P < 0.05.
T. Fukutomi et al.
598
RESULTS Patients In 42 patients with PBC, 28 patients were asymptomatic and 14 were symptomatic. In the patients with a-PBC, 15 patients were Scheuer's stage I, nine patients were stage II/III. In patients with s-PBC, four patients were stage I, five patients were stage II/III and three patients were stage IV. As shown in Table 1, total bilirubin levels of the patients with a-PBC and s-PBC were 0.6 f 0.2 mg/dL and 4.3 f 4.4 mg/dL, respectively. Albumin levels were 4.1 f 0.3 g/dL in patients with a-PBC, and 3.6 f 0.5 g/ dL in patients with s-PBC. The AMA positive rate was 79% in patients with a-PBC and 88% in patients with s-PBC. Other clinical data were shown in Table 1.
.r
b
*I
7
Serum levels of the 7s domain of type IV collagen In healthy controls serum levels of the 7 s domain of type IV collagen were 4.28 ng/mL (3.88-4.72 ng/mL) and upper limit of the reference based on the mean + 2 s.d. was 5.20 ng/mL (Fig. 1). Serum levels of the 7 s domain of type IV collagen were 5.97 ng/mL (5.07-7.02 ng/mL) in patients with CAH, 8.23 ng/mL (6.40-10.58 ng/mL) in patients with LC, 6.79 ng/mL (4.76-9.67 ng/mL) in patients with PBC, 5.83 ng/mL (4.55-7.48 ng/mL) in patients with a-PBC, and 9.18 ng/mL (6.53-12.91 ng/ mL) in patients with s-PBC. Elevated serum levels of the 7s domain of type IV collagen were observed in eight patients with CAH (80%), and all patients with LC (100O/0), 34 patients with PBC (8l%), 20 patients with a-PBC (71%) and all patients with s-PBC (1009'0). Patients with LC, PBC, a-PBC and s-PBC had higher levels of serum 7s domain of type IV collagen than healthy controls (P < 0.001, P < 0.001, P < 0.01, P < 0.001,
Cont 10
Mean (ng/rnL) Range (ng/mL)
4
28
CAH
LC
10
10
42
5 97
823
6 79
3 88 4 7 2
PBC
6 40-10 5 8 5 07-7 02
a-PBC 28 583
s-PBC 14
9 18
4 5 5 - 7 40 4
16-9 5 7
6 5 3 - 1 2 91
Figure 1 Serum levels of 7 s domain of type IV collagen (7s collagen) in controls and various liver disease. Cont = healthy female controls; CAH = chronic active hepatitis; LC = liver cirrhosis; PBC = primary biliary cirrhosis; a-PBC = asymptomatic PBC; s-PBC = symptomatic PBC. Range indicates mean - s.d. to mean + s.d. Horizontal bars indicate mean f s.d. and horizontal broken lines show the upper limit of reference (mean + 2 s.d.). Asterisks denote the significant differences between two groups (**P
LIVER A N D BILIARY
Clinical significance of the serum levels of the 7s domain of type IV collagen in patients with primary biliary cirrhosis T. F U K U T O M I , * S. S A K A M O T O , * H. I S O B E , t H. SAKAI,* A. M A S U M O T O * A N D H. NAWATA* *Third Department of Internal Medicine, Kyushu University, Fukuoka, Japan and tDepartment of Internal Medicine, Fukuoka Saiseikai Hospital, Fukuoka, Japan
Abstract The serum levels of the 7 s domain of type IV collagen were measured with a radio-immunoassay in 42 patients with primary biliary cirrhosis (asymptomatic: n = 28; symptomatic: n = 14), 10 patients with chronic active hepatitis, 10 patients with liver cirrhosis and 10 healthy female controls. Serum levels of the 7 s domain of type IV collagen were: 4.28 ng/mL (3.88-4.72 ng/mL; mean and range of mean k s.d.1 in healthy controls; 5.97 ng/mL (5.07-7.02 ng/mL) in patients with chronic active hepatitis; 8.23 ng/mL (6.40-10.58 ng/mL) in patients with liver cirrhosis; and 6.79 ng/mL (4.76-9.67 ng/mL) in patients with primary biliary cirrhosis. Patients with liver cirrhosis and primary biliary cirrhosis had higher levels of serum 7 s domain of type IV collagen than healthy controls (P < 0.001, respectively). Serum levels of the 7 s domain of type IV collagen in patients with asymptomatic primary biliary cirrhosis, 5.83 ng/mL (4.55-7.48 ng/mL) were significantly lower than those in symptomatic primary biliary cirrhosis, 9.18 ng/ m L (6.53-12.91 ng/mL; P < 0.001). Serum levels of the 7 s domain of type IV collagen increased significantly along with advancement of the histological stages of primary biliary cirrhosis. Serum levels of the 7 s domain of type IV collagen in the paired sera of eight patients with asymptomatic primary biliary cirrhosis (mean interval 30 months, range 12-48 months) showed significant rises during the intervals ( P < 0.05), while serum levels of albumin and total bilirubin did not change significantly during these intervals. It is concluded that serum levels of the 7 s domain of type IV collagen were good markers of fibrosis and may be useful for evaluation of the clinical status in the patients with primary biliary cirrhosis. Key words: 7 s domain of type IV collagen, primary biliary cirrhosis.
INTRODUCTION Hepatic fibrosis develops as a consequence of chronic inflammation and hepatocyte necrosis in the liver. An excessive extracellular matrix deposition during the development of fibrosis has been demonstrated in several chronic liver diseases.’-4 Type IV collagen is part of the extracellular matrix which forms the core of the basement membranes. In normal human liver, type IV collagen is distributed in the basement membranes of blood vessels, bile ducts and ductules and around nerve axons. It also exists in the perisinusoidal space, but does not exist in hepatocytes or in most of the interstitial matrix of the portal fields.’-4 Electron microscopy shows that sinusoids in normal human liver lack typical basement membranes, although type IV collagen is discontinuously demonstrated by immunohistochemical study.5 The lack of basement membranes in sinusoids is considered to allow intimate contact between the blood
and parenchymal cells. However, in fibrotic liver, type IV collagen with laminin becomes so prominent in the perisinusoidal space that it develops true basement membrane^,^-^ a process known as a capillarization of s i n ~ s o i d sThis . ~ process interferes with the interchange of material between the blood and the hepatocytes and causes hepatic dysfunction. Type IV collagen is organized in a network fashion; four amino terminal ends of type IV collagen are linked together to form a highly protease resistant structure that is known as the 7 s domain. Two carboxyterminal ends of type IV collagen are combined to form Non-Collagenous Domain 1 (NC 1).6 Recently several radio-immunoassays have been developed that detect the 7 s domain and N C 1 in serum.’-* Elevated serum levels of the 7 s domain and N C 1 were reported in patients with chronic liver disease and it has been proposed that serum levels of type IV collagen are a good marker of hepatic fibrosis.’-’* But in patients with primary biliary cirrhosis (PBC) there has been only one report on serum levels of the 7 s domain of
Correspondence: Shigeru Sakamoto, Third Department of Internal Medicine, Kyushu University, Maidashi 3-1-1, Fukuoka 812, Japan. Accepted for publication 13 June 1992.
Serum levels of the 7s domain of type IV collagen type IV c01lagen.l~The clinical significance of serum levels of the 7 s domain of type IV collagen in PBC has not been established. In this study we measured the serum levels of the 7 s domain of type IV collagen with a radio-immunoassay using polyclonal antibody to the 7 s domain of type IV collagen purified from human placenta in 42 patients with PBC. Its clinical implication in PBC was also evaluated.
597 method. Briefly, assay procedures were as follows: 100 pL of rabbit polyclonal anti-7S domain of type IV collagen antibody (30 000-fold dilution) was added to 200 p L of serum and incubated at room temperature for 16 h. Then 100 p L of [‘251]-labelled 7 s domain of type IV collagen was added, and incubated at 37°C for 2 h. Free and bound antigens were separated by goat anti-rabbit IgG with polyethylene glycols as sediment retainers. The radioactivity in the pellet was counted. T h e intra- and inter-assay coefficient variations were 4.2 and 6.5%, respectively.
PATIENTS A N D METHODS Patients
Laboratory tests
Forty-two patients (female: n = 41; male: n = 1; age range: 30-78 years; mean age: 54 years) who fulfilled established clinical, biochemical, immunological and histological criteria for the diagnosis of PBC were chosen for this ~ t u d y . ’ ~ Twenty-two -’~ were inpatients and 20 were outpatients. Patients with jaundice and/or pruritus were classified symptomatic PBC (s-PBC), whereas patients lacking those symptoms were classified asymptomatic PBC (a-PBC). The patients who were complicated with collagen disease (rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus) known to raise serum levels of the 7 s domain of type IV collagen independently, or had been treated with anti-fibrotic agents (glucocorticoids, colchicine), had positive tests for (HBsAg) or anti c-100, were not included in this study. The patients with increased serum levels of creatinine were also excluded. Ten patients with chronic active hepatitis (CAH), 10 patients with liver cirrhosis (LC) and 10 healthy subjects were chosen as controls. They were all female and age-matched. Diagnosis of CAH and LC was done from biochemical and histological findings. Six patients with CAH and three patients with LC were type B, and four patients with CAH and seven patients with LC were type C.
Serum albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), y-glutamyl transpeptidase (y-GTP), total bilirubin (T.Bi1) and cholesterol were measured by autoanalyser, Toshiba biochemical analyser (Toshiba, Tokyo, Japan). Serum IgM levels were analysed by nephelometry and antimitochondrial antibody (AMA) was detected by indirect immunofluorescence.
Blood sampling Sera were obtained from all patients and controls and stored at - 20°C until assayed. In eight patients with a-PBC, sera were taken at initial diagnosis and during follow-up consultation. The mean follow-up intervals of these eight patients was 30 months (range 12-48 months). These patients remained asymptomatic during these intervals.
Type IV collagen assay The serum levels of the 7 s domain of type IV collagen were determined with commercially available RIA kits (Type IV collagen 7 s kit, Nippon DPC Corp., Tokyo, Japan) according to the manufacturer’s instructions. Details of this assay have been ”uescribed in a previous publication.I2 The 7 s domain of type IV collagen used in this assay was isolated from human placenta and purified as described previously.16 T h e 7 s domain of type IV collagen was iodinated according to the lactoperoxidase
Liver histology Percutaneous liver biopsy with a Tru-cut needle was performed in 36 of 42 patients with PBC for diagnosis. Tissue sections were stained with hematoxylin-eosin and examined by pathologists in our hospital. Histological grading was done according to Scheuer’s classification. Stage I1 and I11 were grouped together because it was difficult to separate them.”
Statistical analysis Logarithmic transformation was performed for statistical calculations to achieve a normal distribution of serum levels of the 7 s domain of type IV collagen. The data in the figures and text are presented as mean and range (mean - s.d. to mean + s.d.). One way analysis of variance (ANOVA) was used to analyse the statistical significance of the differences of the mean levels of serum 7 s domain of type IV collagen between four groups (controls, CAH, LC, PBC) or three groups (controls, a-PBC, s-PBC). Significance was then tested by the Bonferroni method. The correlation between the 7 s domain of type IV collagen and histological stages of PBC was analysed using Pearson’s correlation coefficient. T h e correlations between the 7 s domain of type IV collagen and the liver function test (ALB, T.Bil, ALT, AST, ALP, and y-GTP) were analysed using Pearson’s correlation coefficient. T o achieve a normal distribution, logarithmic transformation was performed on serum T.Bi1, ALT, AST, ALP, and y-GTP before the statistical calculation. The data on serum ALB were not transformed because they were already normal in distribution. T h e significance of the change of the serum levels of the 7 s domain of type IV collagen during follow-up was anal ysed using the Wilcoxon signed-rank test. All tests were considered statistically significant at P < 0.05.
T. Fukutomi et al.
598
RESULTS Patients In 42 patients with PBC, 28 patients were asymptomatic and 14 were symptomatic. In the patients with a-PBC, 15 patients were Scheuer's stage I, nine patients were stage II/III. In patients with s-PBC, four patients were stage I, five patients were stage II/III and three patients were stage IV. As shown in Table 1, total bilirubin levels of the patients with a-PBC and s-PBC were 0.6 f 0.2 mg/dL and 4.3 f 4.4 mg/dL, respectively. Albumin levels were 4.1 f 0.3 g/dL in patients with a-PBC, and 3.6 f 0.5 g/ dL in patients with s-PBC. The AMA positive rate was 79% in patients with a-PBC and 88% in patients with s-PBC. Other clinical data were shown in Table 1.
.r
b
*I
7
Serum levels of the 7s domain of type IV collagen In healthy controls serum levels of the 7 s domain of type IV collagen were 4.28 ng/mL (3.88-4.72 ng/mL) and upper limit of the reference based on the mean + 2 s.d. was 5.20 ng/mL (Fig. 1). Serum levels of the 7 s domain of type IV collagen were 5.97 ng/mL (5.07-7.02 ng/mL) in patients with CAH, 8.23 ng/mL (6.40-10.58 ng/mL) in patients with LC, 6.79 ng/mL (4.76-9.67 ng/mL) in patients with PBC, 5.83 ng/mL (4.55-7.48 ng/mL) in patients with a-PBC, and 9.18 ng/mL (6.53-12.91 ng/ mL) in patients with s-PBC. Elevated serum levels of the 7s domain of type IV collagen were observed in eight patients with CAH (80%), and all patients with LC (100O/0), 34 patients with PBC (8l%), 20 patients with a-PBC (71%) and all patients with s-PBC (1009'0). Patients with LC, PBC, a-PBC and s-PBC had higher levels of serum 7s domain of type IV collagen than healthy controls (P < 0.001, P < 0.001, P < 0.01, P < 0.001,
Cont 10
Mean (ng/rnL) Range (ng/mL)
4
28
CAH
LC
10
10
42
5 97
823
6 79
3 88 4 7 2
PBC
6 40-10 5 8 5 07-7 02
a-PBC 28 583
s-PBC 14
9 18
4 5 5 - 7 40 4
16-9 5 7
6 5 3 - 1 2 91
Figure 1 Serum levels of 7 s domain of type IV collagen (7s collagen) in controls and various liver disease. Cont = healthy female controls; CAH = chronic active hepatitis; LC = liver cirrhosis; PBC = primary biliary cirrhosis; a-PBC = asymptomatic PBC; s-PBC = symptomatic PBC. Range indicates mean - s.d. to mean + s.d. Horizontal bars indicate mean f s.d. and horizontal broken lines show the upper limit of reference (mean + 2 s.d.). Asterisks denote the significant differences between two groups (**P
