Clin Biochem, Vol. 25, pp. 467-470, 1992 Printed in the USA. All rights reserved.

0009-9120/92 $5.00 + .00 Copyright ©1992 The Canadian Society of Clinical Chemists.

Clinical Significance of Serum 7S Collagen in Various Liver Diseases SADAKO YAMADA, 1 TAKEAKI SUOU, 1 HIRONAKA KAWASAKI, 1 and NORITAKA YOSHIKAWA2 1Second Department of Internal Medicine, Tottori University Faculty of Medicine, 86 Nishi-Machi, Yonago 683 and 2Nippon DPC Corporation, Ichihara 290, Japan Serum type IV collagen fragment (7S collagen domain) was measured in 30 controls and 152 liver disease patients with a radioimmunoassay using a polyclonal antibody to human placenta 7S collagen. The serum concentrations of 7S collagen (mean ± SD) were 4.2 _+ 0.9 ng/mL in controls, 5.1 -- 2.0 ng/mL in acute hepatitis, 6.5 -- 2.5 ng/mL in chronic inactive hepatitis, 9.5 +- 3.8 ng/mL in chronic active hepatitis, 14.4 ± 7.5 ng/mL in liver cirrhosis, and 14.4 -- 6.9 ng/mL in hepatocellular carcinoma. In acute hepatitis, 7S collagen was slightly increased, whereas type III procollagen N-peptide and prolyl hydroxylase were markedly increased. In chronic liver disease, 7S collagen concentrations increased with the severity of the disease, and also reflected the degree of fibrosis. The serum 7S collagen concentrations were significantly correlated with those of type Ill procollagen N-peptide and prolyl hydroxylase in all subjects. These results suggest that serum 7S collagen concentration is a useful diagnostic aid for determining hepatic collagen metabolism in liver diseases.

brane collagen (8). Indeed, urinary hydroxylysine (Hyl) excretion is increased in chronic liver disease and the u r i n a r y Hyl/Hyp ratio is significantly higher in cirrhosis (9). 7S collagen is the major crosslinking domain of type IV collagen, and is not cleaved off during the assembly of type IV collagen. Thus, serum concentrations of 7S collagen may well reflect hepatic fibrosis better than that of P-III-P. The present study determined the serum concentrations of type IV collagen fragment (7S collagen domain) and evaluated the relationship between 7S collagen concentrations and those of prolyl hydroxylase and P-III-P in various liver diseases.

KEY WORDS: 7S collagen; type IV collagen; liver disease; prolyl hydroxylase; hepatic fibrosis.

SUBJECTS

Introduction

ncreased metabolism and/or deposition of hepatic .collagen are found in acute and, especially in Ichronic liver diseases (1-3). Several noninvasive methods have been developed for evaluating hepatic collagen metabolism using serum samples. Hydroxyproline (Hyp)-containing peptide, type III procollagen N-peptide (P-III-P), prolyl hydroxylase, and lysyl oxidase activity have been found to be increased in liver disease (4-7). In acute hepatitis (AH) and chronic active hepatitis (CAH), elevated P-III-P values are generally seen in patients without fibrosis, as examined by light microscopy (5). Serum P-III-P concentration therefore seems to be related to fibrogenesis, and not to fibrosis in the liver. In advanced liver disease such as cirrhosis, the sinusoid may develop a basement membrane composed of type IV collagen and laminin. Antigens related to the basement membrane are present in the circulation, and are considered to reflect the metabolism and/or deposition of hepatic basement mem-

Correspondence: Dr. Sadako Yamada. Manuscript received January 29, 1992; revised May 6, 1992; accepted May 8, 1992. C L I N I C A L B I O C H E M I S T R Y , V O L U M E 25, D E C E M B E R 1992

Materials and methods

We examined 152 patients with liver disease and 30 healthy controls after obtaining informed consent to participate in this study. The patients consisted of 28 with AH, 16 with chronic inactive hepatitis (CIH), 25 with CAH, 53 with liver cirrhosis (LC), and 30 with hepatocellular carcinoma with cirrhosis (HCC). Their laboratory findings are shown in Table 1. The mean age of the control group was different from those for LC and HCC. However, in adult subjects, there is no age effect on normal ranges for fibrous markers such as P-III-P, prolyl hydroxylase and lysyl oxidase. Diagnosis was confirmed by histological, immunological and laboratory evaluation. Hepatic fibrosis was graded on a scale of 0 to IV by two observers (NM and YH), according to the histology activity index reported by Knodell et al. (10). Serum samples were separated by centrifugation at 4 °C and stored at - 4 0 °C until analyzed. The concentration of 7S collagen was determined by a radioimmunoassay (RIA) using a polyclonal antibody to 7S collagen isolated from h u m a n placenta type IV collagen, according to the method of Risteli et al. (11). Briefly, the purified antigen was labelled w i t h 125I (Amersham Co., Arlington Heights, IL, USA) by the chloramine T method, to a specific radioactivity of 20,000 counts/min per ng. The primary antibody was rabbit antiserum to 7S collagen and the secondary antibody was goat-antirabbit IgG an467

YAMADA, SUOU, KAWASAKI, A N D Y O S H I K A W A TABLE 1 Clinical and Laboratory Findings in Patients With Liver Disease Hepatitis

Group

Control

No. of cases Age (years) Sex (M/F) Liver function tests A l b u m i n (g/L) ~ - G l o b u l i n (g/L) T o t a l b i l i r u b i n (~mol/L) A S T (IU/L) A L T (IU/L)

35

30 -+ 1 17/13

42.4 12.2 9.4 18.6 20.8

-+ 0.3 - 0.2 -+ 0.5 -+ 0.3 -+ 0.8

Chronic Inactive

Acute 28 -+ 4 16/12

38

41

41.2 -+ 1.0 13.4-+ 0.8 129.0 -+ 9.1 614.2 +- 43.3 725.1 -+ 37.0

Chronic Active

16 -+ 4 9/7

42.1 + 14.7 -+ 14.0-+ 87.1 -+ 118.3 -+

44

1.2 1.3 1.7 15.5 10.4

Liver Cirrhosis

25 -+ 6 18/7

40.3 17.7 17.1 153.2 282.5

59

-+ 0.7 -+ 0.9 -+ 1.2 -+ 27.8 -+ 80.6

53 -+2 33/20

32.2 23.2 36.1 87.3 59.2

-+- 0.8 +- 1.3 -+ 7.4 -+ 7.1 - 5.9

Hepatocellular Carcinoma

61

30 -+ 3 18/12

32.0 20.1 30.8 102.1 84.3

-+ -+ -+ -+

0.9 1.1 5.5 19.1 17.7

Values are mean - SEM.

tiserum. To 200 ~L of serum was added 100 ~tL of primary antibody (diluted 30,000 fold) and incubated at 37 °C for 2 h, then 100 ~L of 12sI-7S collagen was added and further incubated at 37 °C for 2 h. The free and bound antigens were then separated by precipitation with 100 ~L of the secondary antibody containing 3.6% (w/v) polyethylene glycol. The dilutions were made with phosphate-buffered saline, pH 7.4, containing 0.1% (w/v) bovine serum albumin. The coefficient of variation of the 12 assays was 6.8% for intra-assay and 4.1% for inter-assay comparison. The serum P-III-P level was determined with a commercial RIA kit (Behringwerke AG, Marburg, Germany), and prolyl hydroxylase was determined with an EIA kit (Fuji Chemical Industries, Ltd., Takaoka, Japan) (6). The results were expressed as mean -+ SD. The significance of differences was assessed using the Student's t test. Correlations were analyzed by the F-test.

Group

Results Figure 1 shows the results of the determination of 7S collagen in the control group and in patients with various liver diseases. Figure 2 shows the serum concentrations of 7S collagen, P-III-P, and prolyl hydroxylase in the same samples from 166 control and liver disease subjects. In the AH group, 7S collagen was slightly increased, whereas P-III-P and prolyl hydroxylase were 3.0 and 2.4 times higher, respectively, than the control values with significant differences (p < 0.01) for both. Among patients with chronic liver disease, the serum concentrations of 7S collagen increased gradually with the severity of the disease, while the serum concentrations of P-III-P and prolyl hydroxylase were markedly elevated at all times. The serum concentrations of 7S collagen were correlated with serum levels of P-III-P and albumin (r = + 0.422, p < 0.01 and r = - 0.417, p < 0.01), but not with prolyl hydroxylase or other liver function

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F i g u r e 1 - - S e r u m c o n c e n t r a t i o n s of 7S c o l l a g e n in v a r i o u s l i v e r diseases. **p < 0.01, *p < 0.05 as c o m p a r e d to control subjects. No.: N u m b e r of cases. D a t a a r e m e a n -+ SD. 468

CLINICAL BIOCHEMISTRY, VOLUME 25, DECEMBER 1992

SERUM 7S COLLAGENIN VARIOUSLIVER DISEASES Prolyl h y d r o x y l a s e (ng/mlJ

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1.00

P - I I I - P { ng/mL)

l.g

7S collagen (ng/mk}

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Figure 2 - - Serum concentrations of 7S collagen, P-III-P and prolyl hydroxylase in liver diseases. **p < 0.01, *p < 0.05 as compared to control subjects. Data are mean +- SD. tests such as total bilirubin, ~-globulin, aminotransferases, alkaline phosphatase, a-fetoprotein (AFP), ~-glutamyl transferase, indocyanine green clearance rate, and prothrombin time. Figure 3 shows the relationship between the se-

rum concentration of 7S collagen and hepatic fibrosis in 65 patients with chronic liver disease. Serum 7S collagen correlated positively with the degree of hepatic fibrosis (p < 0.01). Discussion

p < 0.01

Figure 3 - - Relationship between serum 7S collagen and hepatic fibrosis in chronic liver diseases.

Basement membrane collagens such as type IV and type V have a higher Hyl content than interstitial collagen. Our previous study showed that hepatic Hyl content was elevated in chronic liver diseases and urinary Hyl excretion was also increased in proportion to the severity of the liver diseases (9). Histological examination showed that fibrotic human liver had true basement membrane consisting of type IV collagen, laminin, and fibronectin (12). These findings suggest that basement m e m b r a n e fragments are present in sera and urine, and m a y reflect metabolism of the hepatic basement membrane. Indeed, serum concentrations of type IV collagen and laminin-related antigens increase in alcoholic liver disease (13). The present study shows that serum 7S collagen concentrations are markedly elevated in cases of chronic liver diseases (p < 0.01 for each group) and gradually increase with the severity of the disease. Also, 7S collagen concentrations are correlated with the degree of hepatic fibrosis (p < 0.01). Thus serum 7S collagen may be a useful index of hepatic fibrosis. P-III-P and prolyl hydroxylase were also studied as indicators of hepatic fibrosis and/or fibrogenesis. In patients with CAH, LC, and HCC, the serum concentrations of 7S collagen correlated with serum levels of P-III-P and prolyl hydroxylase. In cases of acute hepatitis, the serum levels of P-III-P and prolyl hydroxylase were markedly increased, while serum 7S collagen was only slightly increased (Figure 2). Serum concentrations of P-III-P and prolyl hy-

CLINICAL BIOCHEMISTRY,VOLUME 25, DECEMBER 1992

469

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range

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YAMADA, SUOU, KAWASAKI,AND YOSHIKAWA droxylase were well correlated with serum aminotransferase activities (data not shown), suggesting t h a t they are released during the active inf l a m m a t o r y p h a s e of the disease. T h u s s e r u m concentrations of P-III-P and prolyl hydroxylase may reflect inflammation and necrosis, and not necessarily fibrosis. In contrast, serum 7S collagen is increased in cirrhotic patients with and without active liver disease. It may reflect the liberation of antigen from previously-existing hepatic basement membrane r a t h e r t h a n as a product of inflammation. Indeed, there was no correlation between the concentration of 7S collagen and serum aminotransferases. Similarly, except for serum albumin, there was no or only weak correlation between 7S collagen and other liver function tests. The value of serum fibrotic markers such as 7S collagen in the prognosis of the fibrotic process is not yet established, and their superiority over routine liver function tests is yet to be demonstrated. Serum concentrations of P-III-P have been reported to be a useful prognostic indicator in primary biliary cirrhosis (14). This study showed t h a t alcoholic LC patients have significantly higher serum concentrations of 7S collagen t h a n nonalcoholic LC patients. Hepatic collagen metabolism is typically increased in rats with experimentally-induced alcoholic liver injury, as well as in p a t i e n t s with alcoholic liver disease (2,3,15). Indeed, several serum markers, such as P-III-P, prolyl hydroxylase, and laminin are elevated to a greater extent in alcoholic t h a n in nonalcoholic liver disease (13,16). In HCC patients with LC, the mean serum concentrations of 7S collagen were similar to those in cases of LC alone (14.4 -+ 7.5 ng/mL and 14.7 - 6.9 ng/mL). These findings agree with previous reports on P-III-P and laminin levels. In this study, serum 7S collagen concentrations correlated weakly with serum AFP (5,17). Our study showed t h a t serum 7S collagen is a better m a r k e r of collagen metabolism, especially its accumulation in the liver, t h a n other fibrotic indexes such as P-IH-P and prolyl hydroxylase.

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Acknowledgements We are grateful to Drs. Yasushi Horie, Naoto Maeda, and Yosuke Kishimoto for their kind assistance. References 1. Murawaki Y, Hirayama C. Hepatic collagenolytic cathepsin in patients with chronic liver disease. Clin Chim Acta 1980; 108: 121-8. 2. Murawaki Y, Kato S, Hirayama C. Hepatic collagen synthesis and degradation in alcoholic and non-

470

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17.

alcoholic liver disease. In: Fujisawa K, Kimura K, Kawase H, eds. Alcohol metabolism and the liver. Vol V. Pp. 162-7. Tokyo: Toyo Shoten, 1986 (in Japanese). Hirayama C, Murawaki Y, Kato S, Yamada S, Koda M. Hepatic collagen metabolism in alcoholic liver disease. In: Oda T, Okuda K, eds. New trends in hepatology. Pp. 14-22. Tokyo: Medical Tosho, 1987. Yamada S, Hirayama C. Clinical significance of serum hydroxyproline-containing peptides with special reference to hyproprotein. Eur J Clin Invest 1983; 13: 129-33. Yamada S, Suou T, Hirayama C. Clinical significance of serum hyproprotein and aminoterminal procollagen-III-peptide in liver disease. In: Hirayama C, Kivirikko KI, eds. Pathobiology of hepatic fibrosis. Pp. 201-8. Amsterdam: Excerpta Medica, 1985. Yoshida S, Bai Y, Muragaki Y, et al. A sandwich immunoassay for prolyl 4-hydroxylase using monoclonal antibody. Clin Chim Acta 1986; 160: 37-46. Sakamoto M, Murawaki Y, Hirayama C. Serum lysyl oxidase activity in patients with various liver diseases. Gastroenterol Jpn 1987; 22: 730-6. Trivedi P, Risteli J, Risteli L, et al. Serum type III procollagen and basement membrane proteins as noninvasive markers of hepatic pathology in Indian childhood cirrhosis. Hepatology 1987; 7: 1249-53. Yamada S, Aoto Y, Suou T, Hirayama C. Urinary hydroxyproline and hydroxylysine excretions in relation to hepatic hydroxyproline content in chronic liver disease. Clin Biochem 1989; 22: 389-93. Knodell RG, Ishak KG, Black WC, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981; 1: 431-5. Risteli J, Bachinger HP, Engel J, Furthmayr H, Timpl R. 7-S collagen: Characterization of unusual basement membrane structure. Eur J Biochem 1980; 108: 239-50. Hahn E, Wick G, Pencev D, Timpl R. Distribution of basement membrane proteins in normal and fibrotic liver: Collagen type IV, laminin and fibronectin. Gut 1980; 21: 63-71. Niemela O, Risteli L, Sotaniemi EA, Risteli J. Type IV collagen and laminin-related antigens in human sera in alcoholic liver disease. Eur J Clin Invest 1985; 15: 132-7. Eriksson S, Zettervall O. The N-terminal propeptide of collagen type III in serum as a prognostic indicator in primary biliary cirrhosis. JHepatol 1986; 2: 370-8. Yamada S, Yamada M, Murawaki Y, Hirayama C. Increase in lipoperoxides and prolyl hydroxylase activity in rat liver following chronic ethanol feeding. Biochem Pharmacol 1990; 40: 1015-9. Torres-Salinas M, Pares A, Caballeria J, et al. Serum procollagen type III peptide as a marker of hepatic fibrogenesis in alcoholic hepatitis. Gastroenterology 1986; 90: 1241-6. Brocks DG, Strecker H, Neubauer HP, Timpl R. Radioimmunoassay of laminin in serum and its application to cancer patients. Clin Chem 1986; 32: 787-91.

CLINICALBIOCHEMISTRY,VOLUME 25, DECEMBER 1992

Clinical significance of serum 7S collagen in various liver diseases.

Serum type IV collagen fragment (7S collagen domain) was measured in 30 controls and 152 liver disease patients with a radioimmunoassay using a polycl...
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