59

Clinica Chimica Acta, 197 (1991) 59-66 Elsevier Science Publishers B.V. ADONIS 000989819100081V

CCA 04945

Collagen peptidase and type III procollagen peptide serum levels in chronic liver diseases Andreas Miiller I, Bruno Krombholz ‘, Gerhard Pott 2, Georg Machnik 3, Riidiger Vollandt 4, Manfred Reinhardt ’ and Dietfried Jorke ’ ’ Department of Internal Medicine, University of Jena, FRG, 2 Marienhospital

of Nordhorn, 3 Institute of Pathological Anatomy, University of Jena, FRG and ’ Working Group Electronic Data Processing. University of Jena (FRG)

FRG,

(Received 17 April 1990; revision received 15 November 1990; accepted 23 November 1990) Key words: Hepatic fibrosis; Chronic liver diseases; Procollagen III; Collagen peptidase

Summary

The concentration of the N-terminal peptide of procollagen III and the activity of collagen peptidase (PZ-peptidase) were measured in sera from 92 patients with chronic liver disease. In patients with liver cirrhosis and chronic hepatitis with transformation of liver structure, high values were found for both variables compared with hepatoses and chronic hepatitis without transformation. The concentration of procollagen III peptide and the activity of collagen peptidase in serum increased with increasing degrees of fibrosis and, even more markedly, with increasing degrees of mesenchymal activity in the liver.

Introduction

In assessing progress of chronic liver disease the degree and activity of the fibroproliferative process are important [l]. Reliable data about the extent of fibrosis are generally only possible by using histological methods for the evaluation of liver biopsy or by laparoscopy. However, several biochemical parameters have been developed as markers of hepatic fibrosis [2,3]. The N-terminal peptide of procollagen III (P-III-P) which enters the blood during the biosynthesis of type III collagen seems to be a reliable marker. High P-III-P concentrations are found in alcohol-related liver disease. and in chronic active hepatitis [4-61. However, the Correspondence .to: A. MiilIer, Klinik fiir Innere Medizin, Friedrich-Schiller-UniversitBt Marx-Allee 101 Jena-Lobeda, o-6902, FRG.

Jena, Karl-

60

activity of collagen peptidase (CP) (EC No. 3.4.99.31) in serum possibly reflects the degradation of collagen [7]. In chronic liver disease with fibrosis the increase in activity of CP seems to be dependent on the degree of liver fibrosis and its activity

PI. Morelli et al. [9] compared the diagnostic value of P-III-P and collagen peptidase (‘collagen peptidase’ and ‘PZ-peptidase’ are synonyms). Related to diagnostic groups, the authors failed to find any correlation between these variables. As the determination of P-III-P concentration in serum depends on the existence of a nuclear-medical laboratory and because the determination is quite expensive with respect to CP determination, in this paper only the value (usefulness) of both parameters was compared and, in addition to several liver diagnoses, also related to the degree of fibrosis and to mesenchymal activity of the liver. Material and methods

The study included 92 patients (52 males, 40 females) with chronic liver disease. Epidemiological and clinical data as well as laboratory values are presented in Table I. The diagnosis of liver disease was established according to commonly used criteria (clinical, laparoscopy and in all cases histological). The liver biopsies were done as blind biopsies according to Menghini with a needle calibre of 1.4-1.6 mm or monitored by an operative laparoscope (MLW Leipzig). The patients were divided into 4 groups, into three classes depending on the degree of fibrosis and three classes with different degrees of mesenchymal activity. None of the patients was taking glucocorticoid therapy or immunsuppressants. The diagnostic group of hepatoses is represented by degenerative parenchymal lesions (metabolic, medicamentous, reactive with other basic diseases, alcohol toxic etc.). There is no evidence for independent chronic hepatitis. A control group consisted of 75 healthy persons (30 males, 18-65 years, 45 females, 20-60 years). Blood from fasted patients was centrifuged (10 min, 1500 x g, room temp.) after clotting. Freezing the serum at - 20°C for up to 5 days did not produce significantly reduced CP activity. The concentration of P-III-P also remained stable. Liver histology

The liver biopsy specimens were fixed in 5% formal&solution (24 h); after paraffin embedding, slices (5 pm) were stained with hematoxilineosin, prepared for iron detection (Berlin Blue reaction), for PAS reaction and connective tissue staining (Goldner [18]). The histological evaluation concerns several structural elements of liver tissue and inflammatory activity. The data were classified in three different grades on a subjective semiquantitative basis [19-221. Serum parameters

Serum P-III-P concentrations, were measured using a commercial radioimmunoassay kit from Behring AG, Marburg, FRG. The activity of collagen peptidase

61

was determined using the Wtinsch and Heidrich pentapeptide [17] PZ-Pro-Leu-GlyPro-D-Arg as a substrate according to Kirsten et al. [lo]. One collagen peptidase unit (CU) is defined as 1 pmol of PZ-Pro-Leu released/l/h. Other biochemical variables including coagulation diagnostics (PT, PTT), bilirubin, serum protein; protein electrophoresis and the activities of transaminases, of y-glutamyl-transpeptidase, alkaline phosphatase, cholinesterase and leucinaminopeptidase were determined according to the methods in the second pharmacopoeia of the former GDR [ll]. Statistical analysis On the basis of three-dimensional contingency tables, correlations were calculated between P-III-P, CP and the degree of fibrosis or the degree of mesenchymal activity. Discriminant analysis provided values of single biochemical variables to differentiate the different classes of fibrosis or mesenchymal activity degree. The diagnostic sensitivity and specificity of CP and P-III-P to distinguish between slight and severe degrees of fibrosis or of mesenchymal activity were calculated [12]. Results

The reference intervals for each of the biochemical variables measured, are shown in Table I. The highest values for P-III-P and CP were found in the patients with liver cirrhosis and chronic hepatitis with transformation. The values in patients with hepatosis and chronic hepatitis without transformation, however, were within the reference interval. Table I also characterizes the patients on the basis of different biochemical variables. A classification of the patients into three groups with different degrees of hepatic fibrosis reveals a dependence on the degree of fibrosis for both P-III-P and CP (Table II). The increase in both variables was even more marked as mesenchymal activity increased (Table III). The calculation of the interdependence between P-III-P and CP yielded a positive correlation with a correlation coefficient r = 0.52 and a probable error of (Y= 0.01 for all 92 patients. The evaluation of three-dimensional contingency tables using Pearson’s X2-test resulted in a correlation between P-III-P and CP in the different classes of fibrosis as well as in those of mesenchymal activity. For the classes of fibrosis degree, the test value, the likelihood ratio x2-value = 22.01 with 3 degrees of freedom and for the classes of mesenchymal activity = 10.77 with 3 degrees of freedom. On the basis of the ‘F-to Enter’ values, linear discriminant analysis with univariate consideration yielded the order of rank of variables with regard to their contribution to discrimination in the classes of fibrosis. See Table V. In the discrimination in fibrosis classes the additional amount of CP for the separating effect of P-III-P is of course small, because of the good correlation between both parameters. With regard to the degree of mesenchymal activity, the order of rank of ‘F to Enter’ values in univariate consideration is presented in Table VI. In multivariate

I

2.9 0.030 0.026 0.35 0.41 0.63

78.9 f 0.53 f 0.20 f 1.00+ 1.06 f 1.66f

0.48* 0.24 8.94+ 1.69 119.2 f 16.2

0.60 0.26

2.4 0.6

35.7 f 15.8 f

0.8Ozt 0.56f

9.5

1.44 0.47

as multiple

0.72* 0.60 13.69+ 6.17 168.6 f 63.4

1.22* 0.03*

31.2 k 5.2 37.6 k33.2 30.9 zt46.1 80.3 f 6.6 0.52 f 0.063 0.22 f 0.0600 1.85* 1.30 1.63* 1.45 5.45* 8.68

38 29-59 71.1 i-14.2

7 24-51 85.6 f

** Liver cirrhosis

groups:

Hepatoses

Diagnostic

* * Values are shown as mean f SD. * * * Values are shown

LAP *** P-III-P CP

APH *** CHE ***

No of subiects Age (years) Prothrombin time (PT) Partial thromboplastin time (PTT) Bilirubin total Bilirubin direct Serum protein Albumins Gamma-Globulins ASAT *** ALAT *** Gamma-CT ***

Variables

TABLE

0.46 0.42

of normal

range.

0.61 f 0.42 9.26* 2.77 130.0 f 34.2

0.76& 0.45 f

33.9 f 3.6 16.0 & 0.1 6.5 + 0 17.2 k 7.2 0.54 f 0.052 0.18 f 0.037 1.38* 0.60 1.33* 0.72 7.28 f 15.75

20 27-56 79.5 *13.1

Chronic hepatitis without transformation

2.95 0.65

3.2 4.5 2.5 6.2 0.043 0.045 1.50 2.93 17.7

l.OOf 1.22 14.63 f 10.84 145.9 k46.3

1.50+ 0.31+

35.7 + 16.7 f 8.5 f 79.6 + 0.51 f 0.20+ 2.38+ 2.49* 9.52 f

27 30-61 76.2 k12.1

Chronic hepatitis with transformation

0.130 -0.370 pmol/sec. up to 15 rig/ml up to 142 CU

up to 4.85 nmol/sec. 1 6 102 - 174 nmol/sec. 1 0 86-159 pmol/sec. 1 1

30-40 sec. up to 16 pmol/l up to 6 nmol/l 65-85 g/l 0.56 - 0.66 0.12-0.20 up to 0.490 j.rmol/sec. up to 0.570 gmol/sec. 1 6 up to 0.580 prnol/sec. 1 0 up to 0.410 pmol/sec. 1

80 - 100%

Reference interval

63

TABLE

II

Serum levels of P-III-P Degree of fibrosis:

*.

and CP at different

**

degrees

1.5 - 3.0

O-1.0 8.65* 1.80 123.3 f 19.1

P-III-P CP * The units of P-III-P

TABLE

of fibrosis 3.5

13.17* 7.69 153.7 f 53.4

16.78 + 10.05 181.4 +73.7

and CP are the same as in Table I. * * Values are shown as mean + SD.

III

Serum levels of P-III-P Degree of mesenchymal

activity:

and CP at different

*’ * *

P-III-P CP * The units for P-III-P

TABLE

degrees

of mesenchymal

activity

absent-slight

moderate

severe

8.47* 2.31 120.1 f 20.2

14.40* 6.86 164.6 + 58.1

26.7 f 14.1 237.0 f 43.0

and CP are the same as in Table I. * * Values are shown as mean f SD.

IV

Comparison of diagnostic severe degrees of fibrosis

sensitivity and specificity of P-III-P and mesenchymal activity

and CP in the differentiation

of slight and

Degree of fibrosis Diagnostic

sensitivity

Diagnostic

CP:

$=63X

g=791

P-III-P:

+=5ow

$=lOo%

CP:

~=lOO%

%=89%

P-III-P:

4=8OW

$=lOO%

Mesenchymal

TABLE Order

specificity

activity

V of rank of variables

with regard

to their contribution

to discrimination

Variable

‘F to Enter’ value (F-statistic with the degrees of freedom 2 and 73)

P-III-P Bilirubin CP LAP PTT PT

6,961 6,349 4,783 4,238 3,770 3,487

in the classes

of ttbrosis

64 TABLE VI Order of rank of variables with regard to their contribution mesenchymai activity

to divination

Variable

‘F to Enter’ value (F-statistic with the degrees of freedom 2 and 83)

CP P-III-P Bilirubin Gamma-GT pn: Fr ASAT Gamma-globulins

20,718 17,432 17,432 9,561 8,869 8,063 6,840 6,205

in the classes of

consideration, discriminant analysis produces a good discrimination in the classes of mesenchymal activity together with the parameters CP, bilirubin and P-III-P. Table IV shows the values of diagnostic sensitivity and specificity of P-III-P and CP in the differentiation of slight and severe degrees of fibrosis and mesenchymal activity.

Discussion Assuming increased collagen synthesis as well as partial acceleration of degradation during the development of hepatic fibrosis, it seems to be understandable that in active fibropro~ferative processes in the liver, both markers of collagen biosynthesis and of degradation are enhanced [13]. One marker of synthesis for collagen - the N-terminal peptide of procollagen III (P-III-P) - is presently the best available method which can provide data on fibrosis producing processes in hepatic tissue on the basis of laboratory blood examination [14]. Histomorphometrically, a highly significant correlation between serum P-III-P and fibrosis content of hepatic tissue could be demonstrated in chronic liver diseases [4,6]. In contrast, collagen peptidase (CP) or PZ-peptidase is a marker of collagen degradation [15]. The role of this enzyme and its natural substrate in connective tissue metabolism are only poorly understood. Probably CP catalyzes the terminal degradation of collagen fragments resulting from the action of various collagenases [7,X]. Hahn et al. [15] described two pH-optima, pH 7.2 and pH 8.0 for collagen peptidase dete~ation using the artificial substrate PZ-pentapeptide 1171. The pH-optimum 8.0 was only found if the enzyme was estimated 8 hours or a longer time after the blood was taken. Because, in our study, sera was frozen immediately after centrifugation we chose the pH-optimum 7.2 for incubation. According to Hahn et al. (151 the behaviour of the enzyme at both pH-optima is the same in all patient groups.

65

In a clinical study (with patients other than those used in this study) it could be shown that CP can differentiate between liver disease with low and those with high degree of fibrosis with satisfactory sensitivity and specificity [8]. Morelli et al. compared the serum levels of P-III-P and CP in various precirrhotic liver diseases [9]. The authors did not succeed in establishing a statistical relation between the two variables when these were related to the different diagnostic groups. As every chronic liver disease includes inactive as well as active types, an attempt was made in this study to divide the patients not only into diagnostic groups but in groups with different degrees of fibrosis (df) and with different mesenchymal activities (ma). As shown in Tables II and III, with increasing fibrosis as well as, even more markedly, with increasing degree of mesenchymal activity, enhancement of serum levels of both variables can be demonstrated. This shows that both P-III-P and CP reflect rather the activity of the fibroproliferative process in the liver than the amount of hepatic collagen. The analysis of three-dimensional contingency tables demonstrated that an established correlation between P-III-P and CP exists for each of the three degrees of fibrosis and of mesenchymal activity. Moreover the determination of both variables in connection with the total bilirubin allows a classification in one of the three classes of fg and ma. The diagnostic validity is also characterized by sensitivity and specificity. For the differentiation between low and high degrees of fibrosis and mesenchymal activity, with the help of the variables P-III-P and CP, sensitivity and specificity can be defined as follows: diagnostic sensitivity =

number of pathological values in high df/ma total number of patients with high df/ma

diagnostic specificity =

number of normal values in low df/ma total number of patients with low df/ma

As can be seen from Table IV, the validity of both variables is really comparable, particularly concerning ma. Since no nuclearmedical laboratory is necessary for the determination of CP, measurement can be done with any UV-photometer and the price is very reasonable in comparison with P-III-P radioimmunoassay, collagen peptidase as marker of proliferative activity can be recommended particularly for follow-up studies of liver diseases. References 1 Review by an international group. Acute and chronic hepatitis revisited. Lancet 1977;ii:914-919. 2 Hahn EG. Blood analysis for liver fibrosis. J Hepatol 1984;1:67-73. 3 Gressner AM. Measurement of connective tissue parameters in serum for diagnosis and follow-up of liver fibrosis. Ann Clin Biochem 1987;24:283-292. 4 Raedsch R, Stiehl A Das Serum-Prokollagen-Typ-III-Peptid als Fibrosemarker bei chronischen Lebererkrankungen. IM .Med 1987;14:84-89. 5 Van Zanten RAA, van Leeuwen REW, Beukers R, Wilson JHP. Procollagen III-peptide levels in alcoholic liver disease and primary biliary cirrhosis. Neth J Med 1988;32:278-284.

66 6 Gabrielli GB, Faccioli G, Casaril M, Capra F, Bonazzi L, Falezza G, Tomba A, Baracchino F, Corrocher R. Procollagen III-peptide and fibronectin in alcohol-related chronic liver disease: correlations with morphological features and biochemical tests. Clin Chim Acta 1989;179:315-322. 7 Morales TJ, Woessner JF. PZ-peptidase from chick embryos. J Biol Chem 1977;252:4855-4860. 8 Mliller A, Zollmann Chr, Ma&n& G, Krombholz B, Reinhardt M, Jorke D. Enzyme activities of collagen peptidase (CP), monoaminoxidase (MAO) and N-acetyl-/3-D-glucosaminidase (P-NAG) as fibrosis marker in chronic liver diseases. Dtsch Z Verdaustoffwechs Krankh 1988;48:27-34. 9 Morelli A, Verdovelli A, Fiorucci S, Angelini GP, Fini C, Palmorini CA, Floridi A. Type III procollagen peptide and PZ-peptidase serum levels in pre-cirrhotic liver diseases. Clin Chim Acta 1985;148:87-95. 10 Kirsten D, Lenich R, Mtiller A, Schaedel H. Untersuchungen zur Aktivitltsbestimmung bei Sarkoidose - em Vergleich zwischen Angiotensinkonvertase und Kollagenpeptidase. Z Ges Inn Med 1984;39:383-387. 11 2. Arzneimittelbuch der DDR Diagnostik und Labormethoden. Akademie-Verlag, Berlin 1987. 12 Fahrmeir L, Hamerle A, eds. Multivariate statistische Verfahren. Berlin, New York: W. De Gruyter, 1984. 13 Gressner AM, Bachem MG. Mechanismen der Fibrogenese bei chronischen Leberentztindungen. Z Klin Med 1989;44:377-385. 14 Rohde H, Vargas L, Hahn E, Kalbfleisch H, Bruguera M, Timpl R. Radioimmunoassay for type III procollagen peptide and its application to human liver disease. Eur J Clin Invest 1979;9:451-459. 15 Hahn 0, Fricke R, Hoffmann-Blume E, Predel K. Die Bedeutung der kollagenpeptidaseAktivitatsbestimmung in der Differentialdiagnose chronischer Lebererkrankungen. Z Ges Inn Med 1974;28:327-331. 16 Harris ED, Krane SM. An endopeptidase from rheumatoid synovial tissue culture. Biochim Biophys Acta 1972;258:556-576. 17 Wtnsch E, Heidrich HG. Darstellung von Prolinpeptiden. III Ein neues Substrat zur Bestimmung der Kollagenase. Hoppe-Seylers Z. Physiol Chem 1963;332:300-304. 18 Romeis B. Mikroskopische Technik. 16. Auflage. Mlinchen Wien: R. Oldenburg Verlag, 1968. 19 Baptista A, Bianchi J, De Groote J, Desmet VJ, Ishak KG, Korb G, Macsween RNM, Popper H, Poulsen H, Scheuer PJ, S&mid M, Thaler H. The diagnostic significance of periportal hepatic necrosis and inflammation. Histopathology 1988;12:569-579. 20 Sciot R, Staessen D, Van Damme B, Van Steenbergen W, Fevery J, De Groote J, Desmet VJ. Incomplete septal cirrhosis: histopathological aspects. Histopathology 1988;13: 593-603. 21 Ma&n& G, Low 0, Gabler U, Mtiller A, Schubert H, Jorke D. Versuche zur experimentellen Fibroplasie mit Zink und Colchizin. II. Morphologische Untersuchungen. Z Gastroenterol Suppl 1990; (in press). 22 Machnik G. Morphological reaction patterns of the human microscopic contribution. Exp Path01 1990;39:123-138.

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