Biochemical markers of hepatic fibrosis.

Clin Biochem, Vol, 24, pp. 219-239, 1991 Printed in Canada. All rights reserved.

0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.

Biochemical Markers of Hepatic Fibrosis MARIO PLEBANI and ANGELO BURLINA Department of Clinical Biochemistry and Center for Biomedical Research, University of Padova, Italy Most liver diseases lead to a pathobiochemical reaction termed liver fibrosis. This is a dynamic process implying different rates of progression or regression. Thus, histological examination of a liver biopsy is essential for a diagnosis but biochemical tests are necessary for assessing the activity of the process and monitoring its evolution. We review the most important constituents of liver connective tissue and the biochemical tests developed for evaluating liver fibrosis. The aminopeptide of type III procollagen is the most widely used parameter: two different radioimmunoassays have been developed with different affinities for the two circulating forms of the molecule. The determination of serum P3P reveals an elevation of blood levels both in acute and chronic liver diseases. In the first, serum P3P is an index of hepatic necrosis and inflammation which correlates with other biochemical parameters. In the second it is an index of active fibrogenesis. Moreover, in primary biliary cirrhosis this parameter is an independent prognostic variable and an important predictor of survival. Other immunoassays exist for different collagen cleavage products, but their clinical value is not established. Laminin and fibronectin are the principal structural glycoproteins in liver. Fibronectin determination does not seem to be of clinical value in liver disease. In contrast, serum laminin correlates with the severity of portal venous pressure in advanced liver disease. Its concentration parallels the severity of varices and may indicate the risk of bleeding. Hyaluronate is a high molecular weight polysaccharide; raised serum concentrations reflect both its increased synthesis by activated fibroblasts and its impaired catabolism by the liver. Thus, it may be useful for evaluating and monitoring the progression of chronic liver disease. The measurement of the activity of prolyl 4-hydroxylase as well as that of lysine oxidase and other enzymes has been proposed, but their clinical value is not sufficiently demonstrated. A panel of tests (e.g., laminin, hyaluronate and the aminopeptide of type III procollagen) seems to be recommended for a biochemical assessment of liver fibrosis in clinical practice.

KEY WORDS: fibrosis; liver biopsy; chronic active hepatitis; alcoholic liver disease; primary biliary cirrhosis; aminopeptide of type HI procollagen; 1Amiuin; hyaluronate; fibronectin; prolyl hydroxylase.

precede liver cirrhosis that is a well-defined pathological state characterized, in addition to extensive fibrosis, by rebuilding of the architecture of lobuli and formation of regenerative nodules (4,5). It is clearly established that hepatic fibrogenesis is an active process (6) in which connective tissue synthesis is stimulated in both mesenchymal and parenchymal liver cells (7) and is one of the most important factors leading to disturbed liver function (8). From a clinical point of view, deposition of collagen in perivenular spaces may be the earliest manifestation of the process which ultimately leads to cirrhosis. Fibrosis can be determined by morphological examination of the liver, but this approach cannot be used to assess accurately the activity of collagen synthesis at any given point in time. Thus, the development of biochemical markers of hepatic fibrosis might allow a promising diagnostic approach for the identification and quantitation of this process (9-11). The serial determinations of biochemical markers of fibrosis may provide a non-invasive dynamic evaluation of the disease. In particular they may provide an objective assessment of the progression of the disease and the monitoring of antifibrotic therapeutic treatment. The aims of this review are to outline the main components of the liver connective tissue and their alteration in liver fibrosis. The usefulness of proposed biochemical indices of fibrosis is described by considering their positive and negative aspects.

Hepatic fibrosis ost chronic liver diseases of different etiologies are accompanied by a pathobiochemical reaction termed fibrosis (1,2). In general, this may be defined as deposition of connective tissue to a higher than normal extent (3). Fibrosis is not synonymous with cirrhosis. In fact liver fibrosis may

M

Correspondence:Dr. Mario Plebani, Department of Clinical Biochemistry, Laboratorio Centrale, Ospedale Civile, Via Giustiniani 2, 35128 Padova, Italy. Manuscript received August 20, 1990; revised January 24, 1991; accepted January 25, 1991. CLINICALBIOCHEMISTRY,VOLUME24, JUNE 1991

Hepatic fibrosis is not a uniform phenomenon and it comprises: (a) increased deposition of the liver connective tissue components in the intercellular space (extracellular matrix component, collagens, elastin, proteoglycans, structural glycoproteins); (b) histologically disordered deposition of hepatic connective tissue elements (mRinly a preferential and early accumulation in the perisinusoidal space), leading to disturbances of intrahepatic blood flow and hindrance of exchange processes between blood and cells (3,10,12,13). The cells responsible for hepatic collagen forma219

PLEBANI AND BURLINA

tion remain disputed at this time. Until recently, it was assumed that collagens are elaborated exclusively.by fibroblasts or fibroblast precursors (14,15). Among the resident cells in normal liver, hepatoc~es, sinusofdal endothelial cells and lipocytes appear to synthesize collagen (16). Hepatic lipocytes (Ito cell.s, fat-sto~ing cells) in primary culture were shown to s.e~rete collagen types I, HI and IV and also laminm. Under certain conditions liver fatstoring cells proliferate strongly and become transformed into myofibroblast-like cells (17). It was shown recently that secretions produced by activated Kuppfer cells and monocytes stimulate the proliferation/transformation of fat-storing cells in culture (18,19), as well as the synthesis of proteoglycans (20) and hyaluronic acid (21). Candidate mediators of macrophage-derived fibrogenic activity are platelet derived growth factor (PDGF), and transforming growth factors ~ and (22-24). Other stimulants of fibrogenesis are interleukin l-a, which is the most potent stimulator of fibroblast-produced collagen, and gamma-interferon (1,25-28). Other lymphokines and monokines produced specifically by T-lymphocytes and macrophages may play a role in collagen formation. Acetaldehyde (29,30) and other metabolites of ethanol oxidation have also been reported to stimulate collagen production in liver fibroblasts. The low selenium values observed in serum of patients with liver disease and their inverse correlation with circulating levels of collagen peptides (31,32) suggest that Se deficiency could accelerate the progression of liver damage by decreasing the protective effect of the Se-containing enzyme glutathione peroxidase (EC 1.11.1.9) against the development of hepatic fibrosis. Hepatic fibrosis should be viewed as a dynamic process (6). This concept implies the existence of different rates of progression or regression of liver fibrosis in patients with chronic liver disease (33,34). Regression of fibrosis has been documented in experimental animals (35) and in humans (36-38), suggesting that this is possible when fibrosis and the nodular transformation of hepatic architecture are not too advanced. Hepatic fibrosis is defined on the basis of histological evaluation; in fact liver biopsy is precise in defining the presence and pattern of excess connective tissue at a given point of time, and histopathology is and will remain the mainstay for the diagnosis of hepatic fibrosis and cirrhosis. However, histologic evaluation represents a static cumulative process, revealing little information about the current collagen-deposition activity. Other less invasive techniques, independent of the patient's general condition and repeatable at short intervals, are necessary in order to have information about the activity (connective tissue build-up in time) and reversibility (connective tissue degraded in time) of the fibrotic process.

220

Connective tissue in Hver

Chemically, the normal liver contains about 5 to 8 mg of collagen per g wet weight. The typical connective tissue proteins (collagens, structural glycoproteins and proteoglycans) are present not only in vessel walls, perivascular areas and in the capsule, but also in small amounts in the parenchyma, mainly in the space of D~sse along the sinusoidal walls (39). Drop-out of parenchymal cells and collapse of the preexisting reticulin framework of the liver result in the formation of slender acellular septa (33). Fibrosis may also be caused by collagen biosynthesis usually associated with hepatic injury. In most cases of hepatic fibrosis, both collapse and increased collagen biosynthesis are involved: of these, increased biosynthesis is probably the more important. COLLAGEN

Collagen, derived from a Greek word meaning "to produce glue," is a heterogeneous class of extracellular proteins (3) characterized by a unique aminoacid composition (about 30% glycine, 20% proline + hydroxyproline and a variable content of hydroxylsine). The content of glycine residues in collagen molecules is unusually high for a protein; furthermore, collagen contains two aminoacids that are found in very few other proteins, namely 4-hydroxyproline and 5-hydroxylysine. Finally, the aminoacid sequence of collagen is remarkably regular: nearly every third residue is glycine. Moreover, the sequence glycine-proline-hydroxyproline recurs frequently. Type I collagen consists of two chains of one kind, termed ~l(I), and one chain of another kind, termed ~2(I). Some types of collagen, such as type II, contain three identical chains (Table 1). Each of the three strands in collagen consists of about a thousand aminoacid residues. Collagens can be roughly divided into two groups: those which form collagen fibers in the extracellular space and those which do not. The first group, comprising types I, II and III, is referred to as "interstitial collagens." The second group of collagens has been collectivized under the term '%asement membrane collagens" although it is not yet clear whether they are all components of basement membranes and whether their occurrence is restricted to basement membranes (Table 1). Detailed information on structure and biochemistry of collagens is presented in reviews (40-45). The fibrotic liver shows an increase in all collagen types irrespective of etiology. However, an enhanced production of type III collagen (and a reduction of the ratio of type I to type III collagen from 2.5-4.0 in the normal liver to 0.8-2.0

CLINICAL BIOCHEMISTRY, VOLUME 24, JUNE 1991

M A R K E R S OF HEPATIC FIBROSIS

TABLE 1 Molecular Heterogeneity and Distribution of Collagen in the Liver

Ratio Ratio Chain Composition Hyp/Pro Hyp/Lys

Type (a)

(b)

I

(Interstitial type)

H III

" "

IV V

[al(I)]2a2(I)

Site

0.7

0.2

[~l(II)]s [~l(I~)] s

0.8 1.2

1.9 0.2

Portal zones, central zones, broad scars Sinusoids Reticulin fibres

[al(IV)]2a2(IV) [al(V)]2a2(V)

2.1 1.0

4.4 1.6

Basement membranes Basement membranes

Hyp = Hydroxyproline; Pro = P r o l i n e ; Lys -- Lysine.

in the fibrotic liver) has been described in this condition (3). GLYCOPROTEINS

Glycoproteins are regularly occurring components of the extracellular matrix of connective tissues (46). This class of proteins is heterogeneous in its chemical nature, as well as in its physiological functions, which include formation of matrices for elastin fibers (47), regulation of collagen deposition as well as matrix interaction (48-50). Recently, the roles of two specific glycoproteins, fibronectin and laminin, have been outlined. Fibronectin is a high molecular weight glycoprotein composed of two similar polypeptide chains (51). Each chain has a molecular weight of approximately 250,000 daltons and the two chains are linked by interchain disulfide bonds. Furthermore, heterogeneity in the size of the fibronectin chains was observed. This heterogeneity is the result of variation in both the aminoacid sequences and posttranslational modifications. The functions of fibronectin depend on its binding to a number of different biological structures, e.g., collagen, heparin, fibrin and cell surface components. Specific domains of fibronectin bind and release collagen fibers. Hence, the cells adhere to the extracellular matrix and, in effect, move along tracks of collagen. The heparin-binding domain enables cells to bind to the glycosaminoglycan component of proteoglycans, other components of the connective tissue (52,53). Laminin is a major noncollagenous structural component of basement membranes (54). It is a very large three-chain protein (1000 KD) with a uniform shape. Like fibronectin, laminin consists of multiple domains with distinct binding functions. It enables epithelial cells to attach to underlying connective tissue. Specifically, it has high affinity for type IV collagen, another component of basement membranes (55,56).

CLINICAL BIOCHEMISTRY, VOLUME 24, J U N E 1991

PROTEOGLYCANS

The connective tissue of the liver is also rich in proteoglycans, which consist of units made of polysaccharide (about 95%) and'prStein (about 5%). These very large polyanions bind water and cations and thereby form the extracellular medium, or ground substance, of connective tissue. Proteoglycans are very important in determining the viscoelastic properties of tissue structures (57,58). Glycosaminoglycans, the polysaccharide chains in proteoglycans, are made up of disaccharide repeating units containing a derivative of an amino sugar, either glucosamine or galactosamine (59). Hyaluronate, chondroitin sulfate, keratan sulfate, heparan sulfate and heparin are the major glycosaminoglycans (60). Fibrosis leads to a considerable enhancement of glycosaminoglycan synthesis and to shifts in the tissue pattern of glycosaminoglycans. Dermatan sulfate and chondroitin sulfate are more prominent in the fibrotic than in the normal liver (61). Despite a lack of studies on serum glycosaminoglycans in patients with liver diseases, serum hyaluronate levels have been proposed as a useful tool in biochemical evaluation of those patients. In fact, this glycosaminoglycan, synthesized by fibroblasts and degraded by the specific hydrolase, hyaluronidase, is removed from the circulation by the liver (62,63). ENZYMES OF COLLAGEN SYNTHESIS

Collagen synthesis involves a number of specific post-translational modifications of polypeptide chains, such as the hydroxylation of certain prolyl and lysyl residues to 4-hydroxyproline, 3-hydroxyproline and hydroxylysine, and glycosylation of some of the hydroxylysyl residues to galactosylhydroxylysine and glucosylgalactosyl-hydroxylysine. These intracellular modifications are catalyzed by specific enzymes (64,65): prolyl-4 hydroxylase

221

PLEBANI AND BURLINA

(EC 1.14.11.2),prolyl-3 hydroxylase (EC 1.14.11.7), lysylhydroxylase (EC 1.14.11.4), galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66), and procollagen galactosyltransferase (EC 2.4.1.50). Other enzymes catalyze the conversion of procollagen to collagen and the formation of intra- and interchain cross-links. A specific enzyme, lysine oxidase (EC 1.4.3.14), is responsible for oxidative deamination of the e-amino groups of lysine and hydroxylysine to form the corresponding S-semialdehydes. These then condense either with the e-aminogr.oups of other lysyl or hydroxylysyl residues to form Schiff bases or aldol crosslinks (66). Finaliy,~collagenases contribute to collagen degradation (67-69). " Bioch'emical m a r k e r s of liver fibrosis / ~ I N O T E R M I N ~ L P R O C O L L A G E N TYPE III PEPTIDE

Currently, the most promising blood analyses for liver fibrosis are the immunological determinations of intact procollagen molecules and/or cleavage products of the maturation pathway of procollagen in serum or plasma.

Biochemistry and physiology Procollagens are synthesized intracellularly and have extension peptides at both amino and carboxy ends of the molecule (70,71). The amino terminal peptides are precursor-specific segments, which are cleaved off by specific proteases and released into the circulation during the conversion of procollagen into collagen (72). Since procollagen peptides are liberated in stoichiometric amounts during the conversion of procollagen into collagen and persist for some time in the body, it has been suggested that this reflects the intensity of connective tissue formation in the liver (73). In particular, the aminoterminal procollagen IH peptide (P3P) determination in serum has been proposed as a useful tool for biochemical assessment of liver fibrosis. In fact, type IH is the predominant collagen type in hepatic fibrosis, and only in late cirrhosis is type I predominant (74-76). The concentration of P3P in the liver vascular outflow (hepatic vein) is significantly correlated with the respective propeptide level in the periphery (cubital vein), the Spearman's rank correlation coefficient being 0.734, although the mean level in the hepatic vein (27.6 ~g/L) was higher than that in the cubital vein (20.4 }xg/L) in all groups of liver diseases (77). Thus, the concentration of P3P from cubital vein serum is a good index of the peptide level in the liver. Up to now little was known about the metabolism of P3P. Bile and urine seem to be the major excretion routes of the molecule. The column chromatographic behavior of the antigenic material in bile and its inhibition curve in the radioim-

222

munoassay (RIA) demonstrate that in bile the complete P3P molecule, Coll_s is excreted. On the other hand, a degradation product of the procollagen peptide, Co11, is excreted in the urine (78). The biliary excretion of the propeptide seems to be quantitatively more important than its renal excretion both in cirrhotics and in healthy controls. The mechanism of biliary excretion is unclear, but the size of the propeptide molecule (Mr = 45,000) suggests a vesicular transport (79).

Assays The original RIA for P3P determination developed by Rohde et al. (80) employs a heterogeneous antigen existing in two immunoreactive molecular sizes (81). One antigen resembles the intact triplestranded aminopeptide in size (Co11_3; Mr = 45,000); the other resembles the globular domain of the peptide (Col1; Mr = 10,000) believed to be a proteolytic degradation product of the intact molecule. Both these immunoreactive forms bind to the intact antibody used in the standard RIA, but Col1_3 has a 10-fold greater binding affinity than Col~ (82). In this RIA (standard RIA), several different dilutions of each serum sample are assayed in duplicate in order to calculate the P3P concentration which causes 50% inhibition of binding. The calculation of the 50% intercept on the standard inhibition curve is necessary in the assay because Co11_3 and Col 1 have different binding affinities for the intact antibodies and produce inhibition curves with different slopes (80). This difference in binding affinity causes imprecision in the quantitative analysis of P3P in biological samples such as blood which contain both immunoreactive forms. More recently a RIA that seems to circumvent this imprecision has been developed (83). In this new assay, intact antibodies are replaced by their monovalent Fab fragments which have equal binding affinity for both molecular forms of P3P. The calculation of the 50% inhibition intercept as performed in the standard RIA is thus not required (82). The Fab radioimmunoassay seems to be the most highly recommended method for the peptide determination, but some authors suggest that the combination of standard and Fab RIAs provides more complete clinical information (84). New synthesis and physiological scission of P3P are measured with the standard RIA, while additional degradation is detected by the Fab RIA. Obviously, different ranges for P3P in serum with the two RIAs are found.

Reference ranges Depending on changes in the rate of Type III collagen synthesis in healthy tissues during growth, very different values are found in the serum of adults and children. Highest values of P3P occur during periods of rapid growth, and the lower adult values are reached when growth is complete in late adolescence.


M A R K E R S O F HEPATIC FIBROSIS 400-

300.

.-I O~ ::L

200.

100

0 1-10 d

1-6 m

6-12 m

1-3 y

3-9 y

9-16 y

16-20 y

AduIl

Figure 1--Reference values of P3P in children and adults (d = days; m = months; y = years). Data from Trivedi P e t al. (85). With the standard RIA, highest values of 298 -+ 88 ~g/L (mean -+ SD) are found in the neonatal period (85). P3P fell to 30.9 -+ 7.0 ~g/L by one year, 19.1 _+4.5 ~g/L by three years and rose significantly at puberty. Adult levels (8.3 -+ 3.2 ~g/L) occur by 16 years of age (Figure 1). With the Fab RIA the reference range for adults is between 26 and 75 ~g/L (86). An upper limit of normal of 75 ~g/L is also recommended by McCullough et al. (87). This value is in good agreement with upper normal limits suggested by other authors (84,88). Serum P 3 P levels are with or near the normal range for age in children with chronic active hepatitis, even when they have biochemical and histological evidence of active liver disease. Thus, the high serum values observed in healthy children seem to mask any increase which occurs in P 3 P due to liver disease activity during childhood. A further complicating factor in children m a y be a fall in serum P 3 P due to decreased growth rate secondary to liver disease or its t r e a t m e n t (85). A c u t e a n d i n f l a m m a t o r y liver disease

Adults with liver disease show significant increases of serum P3P (87). Annoni et al. (89) observed elevated P3P levels in serum of patients with acute viral hepatitis and this increase was in line with the pattern of serum aminotransferase activity. This suggests that P3P m a y reflect hepatic necrosis and inflammation. A similar correlation between P 3 P serum levels and aspartate aminotransferase (AST) or other biochemical markers of inflammation was found by some groups (90-93), while others did not observe any relationship between these parameters (80,9498). Bentsen et al. (99) found high P3P serum levels in all patients with acute viral hepatitis; the peptide became normal within 6 months in 91% of


patients with uncomplicated hepatitis; on the other hand, from the second month of the follow-up and throughout the observation period the P 3 P serum concentration was significantly higher in patients who developed chronic disease, as compared to the group with uncomplicated disease (99,100). A possible explanation of the correlation between serum P 3 P and biochemical indices of liver inflammation m a y derive from the recent immunoelectron microscopic demonstration of the persistence of extension aminopropeptides of type HI procollagen in collagen fibrils of the normal liver (101103). Therefore, the propeptides in serum derive not only from newly synthesized procollagen b u t also from mature collagen tissue and could represent the results of both increased fibrogenesis and release during the inflammatory process. Moreover, this observation m a y explain the relationship between serum P3P and both portal inflammation and focal intralobular necrosis and degeneration. Chronic liver disease

Frei et al. (104) measured serum P 3 P (Co11_8) in 111 patients with chronic liver disease, and in 60 patients the P3P values were correlated with liver histology and morphometry. They found that Coll_~ concentrations were significantly elevated in patients with untreated chronic active hepatitis, cirrhosis and primary biliary cirrhosis, but not in chronic persistent hepatitis or fatty liver. A significant correlation between morphometrically measured portal tract area and Co11_3 plasma levels was found, as well as between the number of fibroblasts and the N-terminal peptide of procollagen type III. A good correlation between P 3 P and the extent of liver fibrosis as determined by biopsy was found by various authors (84,105-108), and is illustrated in Figures 2 and 3. More interestingly, Weigand et al. (109) described the clinical usefulness of serial determinations of P3P in the follow-up of patients with chronic liver disease, particularly in chronic active hepatitis. Similar results were reported by other authors (86,110). Persistent elevation of the m a r k e r in serum suggests ongoing fibrosis and the development of cirrhosis; on the contrary, normalization suggests remission. P 3 P levels were significantly raised in patients with chronic alcoholic hepatitis (94,98,111, 112,113) and the rate of increase was higher in severe than in mild fibrosis. A practical usefulness was claimed by Fab-P3P determination in discriminating alcoholics with liver fibrosis from those with only fatty liver (87-90). Moreover, serial determination of serum P3P concentrations are useful to identify patients with alcoholic hepatitis pmgree~qng to cirrhosis and for monitoring the severity of the disease (80,98,105). McCullough et al. (87) did not find a significant difference in serum levels of the peptide between chronic active hepatitis and biopsy-proven cirrho-

223

PLEBANI AND BURLINA

80

30

60

/ .J

20

::L

:::L

o_"

a:

,-, .6

O3

(3.

40

LL

10 20

÷

+÷

÷++

+

Degree of fibrosis

Figure 2--Relationship between serum P3P by the standard RIA and the degree of liver fibrosis by histopathology. Data from Sato S e t al. (84).

sis. P3P levels correlated with the clinical stage of the disease (untreated active hepatitis vs. disease in remission) rather than the extent of established fibrosis and cirrhosis. In our experience, we did not find higher values of P3P in liver cirrhosis in comparison to chronic active hepatitis. On the other hand, it was possible to demonstrate a statistically significant difference between patients with an active form of liver disease and those with an inactive form (Figure 4). In fact, sera from patients with inactive liver cirrhosis have lower P3P levels than patients with active liver cirrhosis, and the same behavior was found in patients with chronic active or persistent hepatitis. As demonstrated by McCullough et al. (87), immunosuppressive therapy (with prednisone alone or in combination with azathioprine) significantly decreases serum P3P levels in both cirrhotic and noncirrhotic patients.

Primary biliary cirrhosis In primary biliary cirrhosis (PBC) a significant correlation was found between serum P3P and the histological stage, the highest levels being found in patients with bridging fibrosis and cirrhosis (Ludwig stages 3 and 4). Using the Cox multivariate analysis, the authors found that the prognostic value of serum P3P is independent of other factors shown to affect survival of PBC patients (114). This observation seems to be important in consideration of the limited prognostic value of the conventional histological staging of the disease which also fails to reflect the response to drug treatment (115). Moreover, P3P has been reported to be useful in distinguishing between PBC and chronic persistent hepatitis (95,109) and in diagnosing PBC per se, but the levels in PBC overlap those in chronic active hepatitis (104,109).

224

++

+++

Degree of fibrosis

Figure 3--Relationship between serum P3P by the FabRIA and the degree of liver fibrosis by histopathology. Data from Sato S e t al. (84).

Nonliver disease Extrahepatic secretion may be an important source of elevated serum P3P in light of reports of increased levels in Paget's disease (116), scleroderma (117), fibrosing pulmonary disease (118122) and rheumatoid arthritis (123-125). Levels may also vary with exercise (126) so that a long and presumably incomplete list of other factors must be excluded before a P3P level can be assumed to reflect hepatic conditions. In effect, studies on procollagen production show that serum P3P levels reflect a complex interaction of collagen secretion, degradation and distribution that is affected by hepatic inflammation, biliary secretion and a variety of extrahepatic factors (28, 79). Due to the hepatic and extrahepatic source of P3P in serum and to possible alterations in propeptide clearance, a strict relationship between collagen deposition and P3P release into serum does not hold, since the latter also accumulates and is degraded intracellularly in liver and extrahepatic tissues (127). Thus, while P3P can provide an estimate of the rate of collagen deposition at a given time, it is highly unlikely that a single measurement can ever provide reliable information on total extracellular collagen. P3P is more a serologic marker of active fibrogenesis than an indicator of the extent of fibrosis, and its serial determination is useful in monitoring the progression to cirrhosis and in assessing the therapeutic efficiency of antifibrogenic drugs. OTHER CLEAVAGEPRODUCTSOF PROCOLLAGENS

Sensitive RIAs are also available for the N-terminal and C-terminal peptides of types I and III


MARKERS OF HEPATIC FIBROSIS

sP-III-P

(ng/mL) 60 50 40 30

:

20

I

"""

w•

10

...... "jw~

CPH

,ee~,

CAH

". . . . . . .

-P a "

:

:

eM.

:

"ram ~Ime

Inactive

Active

CHRONIC HEPATITIS CIRRHOSIS Figure 4--Serum P3P in chronic hepatitis and liver cirrhosis. From Diodati Get al. (86). procollagens (128-131). In late cirrhosis, type I collagen is the predominant type (74) and this may explain the normal values of procollagen type III in those patients (84). Therefore, m advanced cirrhosis measurement of a propeptide of type I collagen, in combination with the type III propeptide, may be more helpful (104). Monoclonal antibodies specific for the N-terminal peptide (132) and C-terminal peptide (133) of type I collagen have been produced. More recently, a reliable RIA for h u m a n type I collagen has been developed with an antibody against a fully processed molecule (134). Type I collagen predominantly increases in cirrhotics, with or without active liver disease, and is less related to liver activity than P3P. Furthermore, it is more increased in the serum of alcoholic patients without cirrhosis than in non-alcoholic patients without cirrhosis, reflecting the progressive weakly reversible fibrosis that characterizes alcoholic liver disease (134). Enzyme-linked immunosorbent assays for urine and serum concentrations of the carboxyterminal domain (NC1) (135-137) as well as for the disulfide-rich cross-linking domains (called 7S collagen) of collagen IV have been described (138-140). However, these assays are not currently employed for liver disease evaluation; they seem to be more useful in other diseases in which an alteration of glomerular basement membrane is described, e.g., insulin-dependent diabetes (141). Experience with tests of serum or urine for free proline, free and peptide-bound hydroxyproline, and acid mucopolysaccharides suggests that all these proposed assays to monitor liver fibrosis are limited by their inability to distinguish inflammation and other forms of injury from fibrosis, and also by lack of organ specificity (10,12).


LAMININ

Biochemistry and physiology Laminin is a major basement membrane glycoprotein that may participate in the attachment of cells to the basement membrane network through noncovalent interaction with type IV collagen, protoheparan sulfate, and some other cell surface structures such as sulfated glycolipids (142-145). In normal liver, laminin is present at sites of typical basement membrane structures around bile ducts, ductules, lymphatic vessels, nerve axons and veins, but absent, as judged by immunofluorescence studies, from the sinusoidal wall, which normally is not surrounded by a basement membrane (13,146-148). During the development of h u m a n (146) and experimental (148) liver fibrosis, laminin becomes deposited in the space of D~sse together with type IV collagen, forming a continuous endothelial basement membrane (148-151).

Assays To overcome the low concentration of laminin in soluble form, a RIA for a specific fragment of the protein (P1) has been developed. In fact, with molecular-sieve chromatography of several serum samples, at least two different size classes of the laminin antigen were demonstrated (152). One had an apparent molecular size comparable to the P1 fragment; the other was larger and may correspond to intact laminin or some oligomeric variant. The pepsinresistant P1 fragment, which originates from the central portion of the cruciform molecule accounts for about one-third of the molecular mass of the protein (153-163).

225

PLEBANI AND BURLINA

A competitive RIA which utilizes monovalent antibody fragments (Fab) against the PI fragment, has been developed for laminin determination in serum. The concentrations are given in [arbitrary] units (U/mL) because an international standard of laminin is not available (164). Reference range a n d values in liver disease

] 81.

~

16.

"r"

I= 14-

E

p

I0 a

In healthy subjects, laminin levels by RIA range between 0.81 and 1.43 U/mL (mean, 1.04 U/mL; SD, _+ 0.20), while in patients with hepatic fibrosis the mean is 1.69 (-+ 0.46) and in cirrhosis it rises to 2.58 (_+ 0.87) U/mL (47,48). Pathologically elevated concentrations of laminin were measured in 66% of patients with liver fibrosis and in 83% of cirrhotic patients (165). The extent of elevation of laminin is dependent on the degree of organ fibrosis (166,167). In particular, increased values of laminin were described in alcoholic liver disease by Niemel~i et al. (139), the highest values being in alcoholic cirrhosis. Kropf et a/. (149) observed significantly higher levels of laminin in serum of patients with hepatic disease in comparison with healthy subjects and patients with nonhepatic diseases. Comparing results for the group of patients with liver disease with those for the reference group consisting of healthy individuals, they obtained values for specificity, sensitivity, and diagnostic efficiency of 0.98, 0.80, and 0.92, respectively. In cirrhotic patients the corresponding values were 0.98, 0.91 and 0.96, respectively. The predictive values of positive and negative test results and the likelihood ratio calculated with healthy subjects as a reference group were 0.97, 0.83 and 39, respectively, in patients with liver diseases. The corresponding results for comparison with a reference group of patients with nonhepatic disease were weaker, being 0.81, 0.80 and 4.3, respectively (149). Linear discriminant analysis, based on logarithms of the laminin concentrations alone, with histology as the classification factor, resulted in the correct allocation of 75% of patients with liver fibrosis and cirrhosis. The misclassified cases were proportionately distributed among the two groups of patients. The inclusion of other biochemical parameters, such as P3P, AST, alanine aminotransferase, gamma glutamyltransferase (GGT) and bilirubin, did not significantly improve the classification. The best set of analytes (laminin and bilirubin) yielded 82% correct classifications. On the contrary, P3P alone allowed only 53% of correct classifications (149). A significant correlation (r = 0.884) between portal venous pressure and serum laminin was found (165) in all cirrhotic and fibrotic patients examined (Figure 5). The correlation was higher in cirrhotic (r = 0.921) than in fibrotic patients (r = 0.716). By contrast, a weak correlation between serum P3P and portal venous pressure in these patients was observed (166). The discriminating power of

226

o c

- .J >

6'

,.5

2

2;s

3;s

scram laminin (U/mL)

Figure 5--Correlation between portal venous pressure and serum laminin in liver cirrhosis (n = 12). r = 0.9206; y -- 5.502x - 3.38. From Gressner AM et al. (165), modified. laminin and portal venous pressure with respect to the presence or absence of esophageal varices has been investigated (167). The concentration of laminin was significantly enhanced in patients with varices and paralleled the severity of varices, diagnosed and staged endoscopically according to Paquet et al. (168). The cut-offvalue for serum laminin of 2.5 U/mL yielded a maximum diagnostic efficiency of 84%, while the maximum diagnostic efficiency of the portal venous pressure was 89% at a cut-off value of I0 units. These results seem to support the view that altered metabolism of basement membrane components is related to portal hypertension and, hence, to the formation of varices (164). The increase of laminin in serum might be a consequence of stimulated synthesis or stimulated turnover of basement membranes in the liver, or both. The formation of basement membranes along the sinusoidal cell layer leads to the capillarization of the sinusoid, which is thought to be an important pathogenetic factor for the development of portal hypertension and the impairment of hepatocellular function in cirrhotic liver (13,150,166). Thus, high levels of serum laminin are prognostic indicators of the severity of liver disease, and may indicate portal hypertension and a risk of bleeding from esophageal varices. FmRONECTn~

Fibronectin (also known as cold-insoluble globulin) is another glycoprotein constituent of basement membranes (169,170) that binds with high affinity to collagens, preferentially to type HI collagen (171). For this reason, fibronectin determinations have been proposed in patients with suspected hepatic fibrosis.


M A R K E R S O F H E P A T I C FIBROSIS

Different values of plasma fibronectin are found according to the method used. In particular, the specificity of the antiserum is crucial in determining the results (172). No correlations between flbronectin levels in serum and histological liver features of alcoholic disease were found (94). Fibronectin decreases in the serum of cirrhotic patients, and the values correlate with other indices of hepatic protein synthesis such as serum albumin (173-176). Thus, decreased serum flbronectin seems to be a consequence of liver functional failure, but other authors have found that serum flbronectin was decreased because of competition with activated coagulation proteins, or overactivity of the spleen, rather than as a consequence of liver impairment (177-179). Figure 6 shows the behavior of fibronectin in serum of controls and in patients with various liver diseases. In patients with primary liver cirrhosis we have confirmed the previously observed high levels of flbronectin (176), b u t the increase of this glycoprotein does not correlate with the severity of the disease or with other biochemical indices (180). Thus, fibronectin measurement does not seem to be of clinical value for the assessment of liver disease, and liver fibrosis in particular. SERUM HYALURONATE

Biochemistry and physiology Serum hyaluronate (or hyaluronic acid) is a high molecular weight polysaccharide which is widely distributed in connective tissues and produced mainly by mesenchymal cells; it enters the blood via the lymph (181). The polysaccharide is rapidly taken up from blood by the liver and is accumulated in the nonparenchymal cell fraction (182,183). The pathophysiological mechanism of hyaluronate increase in serum is not completely understood. The most probable hypothesis is that hyaluronate levels in serum reflect both increased synthesis by activated hepatic flbroblasts in fibrotic areas, and the reduced catabolism caused by hepatic failure (184). The second mechanism is confirmed by the correlation between hyaluronate in serum and other markers of hepatic metabolism, and by the remarkable increase of hyaluronate in hepatitis or cirrhosis. In particular, only endothelial cells b u t not hepatocytes or Kuppfer cells seem to be able to accumulate and degrade hyaluronate (185). Electron microscopic autoradiographic studies with radioactive hyaluronate injected intravenously in the rat gave new information about the catabolism of hyaluronate, pointing to impaired cellular uptake as an important cause of its increase in serum (186,187).

Assays and reference ranges RIAs for hyaluronate were developed in 1980 (188,189) and have since been improved (190). By


the last method, the concentrations of hyaluronate in healthy adults were in the range of 10 to 100 ~g/L, with a mean of 35 ~g/L (190). Higher hyaluronate concentrations were noted in older people, but no notable sex difference was found. Similar values in healthy subjects were observed using an enzymoimmunoassay (184).

Chronic liver disease Significantly increased hyaluronate was found in patients with liver diseases (Table 2). Significantly increased serum values were found in chronic active hepatitis (mean values = 282.8 }xg/L) (191); on the contrary, no significant increase was noted in noncirrhotic alcoholic liver disease, chronic persistent hepatitis and drug-induced liver reaction (184). Significantly (p < 0.001) higher levels of hyaluronate were found in cirrhotics in comparison to noncirrhotic patients, all cirrhotic patients showing values exceeding 100 ~g/L, and in most cases (42 of 47) exceeding 200 ~g/L (191). Statistical analysis of the data showed positive correlations between serum hyaluronate concentration and several liver function tests (galactose tolerance test, serum prothrombin time, serum albumin, serum bilirubin, AST). The same authors evaluated the combination of tests resulting in the best prognostic power using linear logistic regression. This procedure showed that the combination of ser u m hyaluronate, alkaline phosphatase (ALP) and galactose tolerance test increases the efficiency to 0.99. Serum hyaluronate increases in PBC merit special emphasis as there is a close relation between these levels and the histopathological changes in the liver, the clinical course of PBC and other serum variables reflecting liver function (192,193). Serum hyaluronate m a y indicate the presence and progress of liver impairment in early and asymptomatic stages of PBC before conventional liver function tests, with the exception of serum prealbumin, become abnormal (192). We have found (194) that hyaluronate concentration is useful to separate early PBC (Stages: I-II) from advanced disease with fibrosis (Stage: III) or cirrhosis (Stage: IV) as shown in Figure 7. In ad-

TABLE 2

Serum Hyaluronate in Liver Disease

Alcoholic cirrhosis Non-alcoholic cirrhosis Primary biliary cirrhosis Primary sclerosing cholangitis

x (~g/L)

Range

467 357 150 66

205-208 114--800

(~g/L)

21-800

19-500

Data from Ref. 191.

227

PLEBANI AND BURLINA 458

40

o o

35 .J "o

o

---

30

v

25

8

o

8

o

8 8

o

o

20 o

15

u_

10

o

0

5,

o

0 a

o 0

i

,

i

i

(d)C

(c)C

CAH

AVH

(n = 6 2 )

(n = 5 8 )

(n = g 8 )

(n = 19)

,

PBC

Controls

(n=21)

(n = 4 0 )

Figure 6--Box plots showing the distribution of plasma fibronectin concentrations in controls and in patients with liver diseases. Boxes cover the middle 50% of the data values, between the 25th and 75th percentiles, the central line being the median. Lines extend out to the minimum and maximum values, but only to those points that are within 1.5 times the box length (interquartile range). The circles indicate extreme points beyond 1.5 times the box length. The following abbreviations have been used: d(C), decompensated cirrhosis; c(C), compensated cirrhosis; CAH, chronic active hepatitis; AVH, active viral hepatitis; PBC, primary biliary cirrhosis. From Ref. 176, modified. vanced cases, serum hyaluronate displays negative correlation with the survival time. All these findings suggest that serum hyaluronate could be helpful in detecting and monitoring hepatic fibrosis and, specifically, PBC. An unknown aspect of the problem is the role of increased hyaluronate levels in contributing to the immunodeficiency observed in patients with cirrhosis. In fact, it has been demonstrated that hyaluronate inhibits phagocytosis (195) and mitogen-induced lymphoblastic transformation (196). ENZYMES: PROLYL 4-HYDROXYLASE

Biochemistry and physiology Prolyl 4-hydroxylase (proline 2-oxoglutarate dioxygenase; EC 1.14.11.2) which catalyzes the 4-hydroxylation of proline residues in procollagen (197, 198) is the rate-limiting enzyme in the synthesis of collagen which is uniquely characterized by its content of the aminoacid hydroxyproline (64). The enzyme is a tetramer (a2~2) composed of two different subunits, a and ~ (199-202). The a-subunit is very easily degraded in the blood while the ~-subunit is stable (203). Immunoreactive prolyl-hydroxylase protein is present in animal (204,205) and h u m a n (206) tissues in two forms: the active enzyme tetramer and an inactive form, the molecular size of which corresponds to that of the enzyme monomer. The latter form is present in liver cells in very large excess over the former (64). The active tetramer in serum constitutes less than 10% of the total enzyme protein. For this reason and because of the presence of inhibitors (207) the determination of prolyl 4-hy-

228

droxylase in blood is difficult. Increases in the activity of the enzyme have been found in experimentally induced fibrosis in the liver (208) and in a variety of other organs (209,210) after exposure to hepatotoxins such as carbon tetrachloride or dimethylnitrosamine (211). Furthermore, a rise in prolyl hydroxylase activity is one of the first changes observed in experimental liver injury even before increases in hydroxyproline content become evident (64).

Assays Clinical experience with prolyl 4-hydroxylase includes the measurement of the enzyme in the liver and in serum. The proline hydroxylase activity in the liver is usually assayed by the method of Hulton et al. (212) with the modified cofactor concentrations and standardization of the assay introduced by Stein et al. (213). This method measures tritiated water formed when tritiated proline present in the collagen substrate is hydroxylated to hydroxyproline. One unit of prolyl 4-hydroxylase has been defined as the amount of enzyme required to synthesize one mole of hydroxyproline per hour at 37 °C under the standardized conditions described. Risteli and Kivirikko (214) described another method for prolyl 4-hydroxylase activity measurement which utilizes 1 4 rC proline-labelled protocollagen prepared in isolated duck embryotendon cells as a substrate (215). However, because of the presence in tissues and in human serum of large amounts of inactive protein immunoreactive to enzyme antibodies, several immunoassays have been developed to determine the total enzyme protein (216,217). First, there was a competitive RIA using a polyclonal antibody. More recently, enzyme immunoas-

CLINICAL BIOCHEMISTRY, VOLUME 24, J U N E 1991

M A R K E R S OF H E P A T I C F I B R O S I S

HYALURONATE

500. 450, 400, 350.

!

300. ,.-1

250. A

--t

200.

15oi ]002 5oi

$ e •

•

---i"

!

•

I-II

III

a~ ¢.

0

IV

HISTOLOGICAL STAGE Figure 7--Serum hyaluronate in different histological stages of primary biliary cirrhosis. From Ref. 194, modified. says and RIAs using a monoclonal antibody have been introduced (218). In comparison to polyclonal antibodies, monoclonal antibodies for prolyl 4-hydroxylase are preferable in various respects, such as stable antibody titer with respect to specificity and reproducibility, and ease of large-scale production (219). Reference range

Contrasting data on prolyl 4-hydroxylase dependence on sex and age are present in the literature. According to Miyabayashi et al. (220), serum prolyl 4-hydroxylase levels vary with age and sex in healthy control subjects, being significantly higher in females (48.5 +_ 13.6 ~g/L) t h a n in males (42.9 _+ 12.0). Highest values were found at age 30. After this age they declined. These changes may be associated with the aging of connective tissue. On the contrary, Kuutti-Savolainen et al. (64) did not observe any variation with age or sex. Liver prolyl 4-hydroxylase

Liver prolyl 4-hydroxylase has been reported to increase in patients with liver diseases such as chronic active hepatitis, liver cirrhosis, PBC and acute viral hepatitis (221-224) and it has been suggested as a useful index of fibroblastic activity in h u m a n liver disease. Hepatic prolyl 4-hydroxylase activity correlates well with the extent of fibrosis and the rate of collagen synthesis in vitro in patients with hepatitis and liver cirrhosis (225,226). Mann et al. (226) demonstrated that liver prolyl 4-hydroxylase acti,rities were raised in all groups CLINICAL BIOCHEMISTRY,VOLUME24, JUNE 1991

NORMAL

STEATOSIS

ALCOHOUC HEPATITIS

EARLY CIRRHOSIS

ADVANCED CIRRHOSIS

Figure 8--Hepatic prolyl hydroxylase activity and histopathological diagnosis in alcoholic patients. From Mann SW et al. (226), modified. of patients with alcoholic liver disease, except those with steatosis, when compared with alcoholic subjects in whom hepatic morphology is normal. The rate of hepatic collagen synthesis in these patients is correlated with the hepatic prolyl 4-hydroxylase activity measured in percutaneous needle biopsy of the liver (Figure 8). In particular, marked rises in prolyl 4-hydroxylase activity over all other groups of alcoholic patients were observed in patients with early cirrhosis, suggesting that the proportion of patients in a state of active fibrogenesis within this group is increased. On the other hand, the enzyme activity in patients with advanced cirrhosis declines, presumably reflecting the decreased fibrogenesis at this stage of the disease where the ratio of fibrous tissue to cells is increased compared with normal. In patients with alcoholic hepatitis the highest activities of liver prolyl 4-hydroxylase are found in those with the most extensive fibrosis on liver biopsy (227). No correlation between enzyme activity and fatty infiltration was observed. A close correlation (r = 0.76, p < 0.001) between hepatic prolyl 4-hydroxylase activity and serum P3P levels was observed in alcoholic patients (98) and the enzyme activities were significantly higher in patients with severe fibrosis t h a n in those with mild fibrosis. However, an attempt to correlate hepatic and se229

PLEBANI AND BURLINA

rum prolyl 4-hydroxylase activity showed no correlation or only a weak correlation (64). A significant decrease in the enzyme activity was demonstrated in patients with alcoholic hepatitis following hospitalization regardless of whether or not they had received corticosteroid therapy (227). On the other hand, the enzyme activity did not serve as a prognostic indicator of hepatic fibrosis in a series of infants and children with various liver disorders (228). Moreover, increased values of the enzyme were also observed in infectious diseases (224).

Serum prolyl 4-hydroxylase As previously reported, the determination of prolyl 4-hydroxylase in serum is carried out by immunoassays because of the presence of inactive forms and inhibitory substances. Serum immunoreactive prolyl hydroxylase measured by monoclonal antibodies has been found to be increased in 55% of patients with chronic active hepatitis, and in 72% of patients with liver cirrhosis (229). Miyabayashi et al. (220) described significantly higher levels of serum prolyl 4-hydroxylase in patients with acute viral hepatitis, chronic active hepatitis, liver cirrhosis and alcoholic liver disease than in healthy subjects. Particularly high values were observed in chronic active hepatitis with piecemeal necrosis and other inflammatory changes, and in active liver cirrhosis. In our experience with an optimized enzyme-immunoassay using monoclonal antibodies, higher levels of prolyl 4-hydroxylase in serum were observed in liver cirrhosis and alcoholic hepatitis, but the overlap of individual values between different groups of patients is a limiting factor in the clinical usefulness of the test (230). A correlation between prolyl 4-hydroxylase levels in serum and the severity of hepatic fibrosis, evaluated histologically (220), as well as with serum P3P (231), was found, although increased levels were also observed in nonflbrotic liver disorders (231). Nagai et al. found elevated prolyl 4-hydroxylase levels in serum of patients with acute hepatitis, hepatocellular carcinoma, metastatic liver neoplasm and cholestatic diseases (232,233). The highest values were observed in patients with cholestatic diseases, and in those patients the prolyl 4-hydroxylase varied concurrently with biliary enzymes (ALP, GGT). Stein et al. observed the same behavior of the enzyme in patients with obstructive jaundice as well as in infectious diseases (234). OTHER ENZYMES

Lysyl oxidase Lysyl oxidase is the enzyme that initiates the biosynthesis of the cross-link in collagen and elas-

230

tin by catalyzing the oxidative deamination of the e-amino groups of certain lysyl and hydroxylysyl residues in these molecules (235,236). Siegel reported the measurement of this enzyme as a useful marker for monitoring changes in collagen biosynthesis (235-237). The rise of lysyl oxidase activity in plasma from rats receiving CC14 corresponds to the activities in the liver. Lysyl oxidase increased approximately 15-fold and prolyl hydroxylase 3-fold (237,238). Some authors observed no measurable activity of the enzyme in serum (228). In other reports, elevated serum values in chronic active hepatitis, PBC and alcoholic liver disease were observed, but the enzyme remained almost normal in chronic persistent hepatitis and fully developed cirrhosis (239). No correlation was found with the histological grade of hepatic fibrosis, or with the concentration of P3P or the activity of monoamine oxidase.

Galactosylhydroxylysyl glucosyltransferase Preliminary data have been presented on serum galactosylhydroxylysyl glucosyltransferase activity (65,214). This increases in patients with primary liver cirrhosis, acute hepatitis and liver metastases. The enzyme activity correlates with serum prolyl hydroxylase and with other markers of cytolysis (AST) or cholestasis (ALP) (65,240).

Monoamine oxidase Another enzyme used for the diagnosis of liver fibroproliferation is monoamine oxidase (EC 1.4.3.4; MAO) (241,246). The functional role of MAO in hepatic connective tissue metabolism is not firmly established, but the enzyme is possibly involved in collagen and/or elastin cross-link formation (247). MAO increases in liver fibrosis in parallel with the progress of hepatic fibrosis (248). Gressner (247) has obtained a high degree of diagnostic specificity for MAO determinations in serum in liver cirrhosis (90-95%) but a sensitivity of only 60%. The predictive value of a positive result at a maximal prevalence of liver cirrhosis of 0.033 is 0.68, if tested against healthy persons, and less than 0.30 if tested against patients with other liver diseases (249). Thus, MAO does not prove useful in the diagnosis of early stages of fibrotic liver diseases, but its elevation indicates advanced fibroproliferation in the liver with high specificity.

N-acetyl- ~-D-glucosaminidase The determination of N-acetyl-[3-D-glucosaminidase (EC 3.2.1.30; NAG), a lysosomal enzyme which catabolizes proteoglycans and glycoproteins, has been suggested as a valuable procedure to indicate liver fibrosis and cirrhosis (250,255). In chronic active liver disease, NAG activity correlates with the severity of fibrosis and with P3P levels (256,257). It was found to have a sensitivity of 52%, specificity of 34%, positive predictive value of 9% and nega-


MARKERSOF HEPATIC FIBROSIS tive predictive value of 98% for hepatic fibrosis in patients with chronic liver diseases (258). NAG activities increase in sera of patients with liver cell necrosis; in contrast, MAO is unaffected under these conditions. Thus, the combined use of both enzymes in evaluating liver cirrhosis and fibrosis has been suggested (259). Ackermann et al. emphasized the clinical usefulness of the combined determination of P3P and NAG for determining the reversibility of hepatic fibrosis; in fact the P3P level permits estimation of the synthesis, and NAG activity indicates the catabolism of connective tissue (260). NAG has also been reported to be elevated in acute hepatitis, diabetes mellitus and arthritic disorders (261-263). Determination of NAG isoenzymes might enhance the degree of diagnostic specificity (264,265), but further studies are necessary to validate these preliminary results. Collagenases and peptidases It was emphasized t h a t collagen breakdown is a multienzyme process, first accomplished by true collagenases and subsequently by other proteolytic enzymes (266-268). Unfortunately, potent naturally occurring circulating inhibitors hinder the measurement of collagenase activity in serum (269). Recently, it has been demonstrated t h a t PZ-peptidase, an endopeptidase t h a t intervenes in the final stages of the collagen degradative pathway (270), is closely correlated with the tissue collagen degradation rate (271,272). The P3P/PZ-peptidase ratio has been proposed as a dynamic non-invasive index of hepatic fibrosis. A low P3P/PZ-peptidase ratio, due to unbalanced increase in PZ-peptidase activity, was demonstrated in chronic persistent liver disease, whereas a raised ratio was observed in chronic active hepatitis (273).

Conclusion The importance of biochemical markers of fibrosis is evident. In fact, if we consider hepatic fibrosis as a dynamic process it is clear t h a t we need non-invasive, widely available probes of fibrotic activity and reversibility. Despite the development of several tests for the m e a s u r e m e n t of collagen-related enzymes and metabolic products of collagen, their clinical usefulness is not unanimously accepted. None of these parameters is organ-specific, although extrahepatic sources of their changes in serum can be frequently distinguished by clinical and laboratory means. In fact, biochemical markers of fibrosis cannot substitute for liver histology and morphometry which will remain the m a i n s t a y for the diagnosis of liver disease. Biochemical tests should be used for monitoring the fibrotic process; thus, for a better utilization of biochemical tests, it is necessary to perform serial and not one-point measurements. The serial deter-

CLINICALBIOCHEMISTRY,VOLUME24, JUNE 1991

mination of collagen products allows both an objective assessment of the progression of fibrogenesis and the monitoring of antifibrotic therapy. Due to the limitations of each biochemical marker, the most promising diagnostic approach seems to be the combination of some parameters. From a theoretical point of view, the better combination might be t h a t between a m a r k e r of fibrogenesis and a marker of connective tissue degradation. At present, we suggest a panel of tests consisting of the aminoterminal peptide of type III procollagen, laminin and hyaluronate. In particular, this biochemical panel is useful for evaluating the progression of alcoholic liver disease which is a common cause of chronic liver disease.

Acknowledgements We thank Professor David M. Goldberg for his valuable advice during the preparation of this review. We are grateful to Alda Giacomini, M.D., who participated in the literature search and to Maria Grazia Marzellan who typed the manuscript.

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MARKERS OF HEPATIC FIBROSIS

57. 58. 59. 60.

61.

62. 63. 64.

65.

66.

67.

68. 69.

70. 71.

72. 73. 74. 75.

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Hepatic fibrosis and its serum markers: a review.

Biochemical and immunochemical study of lysyl oxidase in experimental hepatic fibrosis in the rat.

[Biochemical markers after trauma].

Biochemical markers of lung disease.

Biochemical Markers of Myocardial Damage.

Liver Shear-Wave Velocity and Serum Fibrosis Markers to Diagnose Hepatic Fibrosis in Patients with Chronic Viral Hepatitis B.

Current status of fibrosis markers.

Noninvasive Markers of Liver Fibrosis.

Therapy for hepatic fibrosis.

Autophagy in hepatic fibrosis.

Congenital hepatic fibrosis.

Biochemical markers in bronchial carcinoma.

Biochemical and immunological markers of over-training.

Biochemical and haematological markers of alcohol intake.

Biochemical and cellular mechanisms of pulmonary fibrosis.

Biochemical and molecular genetics of cystic fibrosis.

Evaluation of biochemical markers in breast cancer.

Biochemical markers of ethanol effects on brain.

Myofibroblasts in hepatic schistosomal fibrosis.

Hepatic fibrosis caused by alcohol.

What is congenital hepatic fibrosis?

Hepatic methotrexate content and progression of hepatic fibrosis: preliminary findings.

Hepatic fibrosis: Concept to treatment.

Hepatic fibrosis--new therapeutic approaches.