- Email: [email protected]
Serum markers of hepatic fibrogenesis in cystic fibrosis liver disease
Journal of Hepatology 41 (2004) 576–583 www.elsevier.com/locate/jhep
Serum markers of hepatic fibrogenesis in cystic fibrosis liver disease Tamara N. Pereira1, Peter J. Lewindon1,4, Jeffery L. Smith1,5, Therese L. Murphy2, Douglas J. Lincoln3, Ross W. Shepherd4,†, Grant A. Ramm1,* 1
The Hepatic Fibrosis Group, The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia 2 Iron Metabolism Group, The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia 3 Cancer and Population Studies Group, The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia 4 Department of Gastroenterology, Royal Children’s Hospital, Brisbane 4029, Australia 5 Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane 4029, Australia
Background/Aims: Hepatic fibrosis contributes to adverse outcome in cystic fibrosis (CF). Early detection of CF liver disease (CFLD) may identify patients at risk of significant complications. To evaluate the utility of serum markers to detect hepatic fibrosis in children with CFLD vs. CF patients without liver disease (CFnoLD) and controls. Methods: Sera from 36 CFLD, 30 CFnoLD and 39 controls were assessed for tissue inhibitor of matrix metalloproteinase (MMP) (TIMP)-1, collagen (CL)-IV, MMP-2, hyaluronic acid (HA) and prolyl hydroxylase (PH) by enzyme immunoassay and were correlated with hepatic fibrosis score in CFLD. Results: TIMP-1, PH and CL-IV were increased in CFLD vs. CFnoLD and controls. Fibrosis score was negatively correlated with TIMP-1 (rZK0.34, PZ0.06) and PH (rZK0.48, PZ0.008). Receiver-operating characteristics analysis showed CL-IV (AUC 0.785, P!0.0001) and TIMP-1 (AUC 0.765, P!0.0001) differentiated CFLD from CFnoLD and controls, while PH (AUC 0.814, P!0.0001) predicted early fibrogenesis. Diagnostic accuracy improved using logistic regression combining (i) CL-IV, TIMP-1, PH to identify CFLD (AUC 0.831, P!0.0001) and (ii) TIMP-1, PH to identify CFLD patients with no fibrosis (AUC 0.852, P!0.02). Conclusions: Elevated TIMP-1, CL-IV, PH may be indicators of hepatic fibrogenesis in CF. Increased TIMP-1, PH may be early markers of CFLD. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis liver disease; Serum markers of hepatic fibrosis; TIMP-1; Prolyl hydroxylase; Collagen type IV
Received 28 January 2004; received in revised form 21 May 2004; accepted 17 June 2004; available online 23 July 2004 * Corresponding author. Tel.: C61-7-3362-0177; fax: C61-7-33620191. E-mail address: [email protected] (G.A. Ramm). Abbreviations: CF, cystic fibrosis; CFLD, cystic fibrosis liver disease; CFnoLD, cystic fibrosis no liver disease; HSC, hepatic stellate cell; PIIIP, procollagen III polypeptide; CL-IV, collagen type IV; PH, prolyl hydroxylase; MMP, matrix metalloproteinase; HA, hyaluronic acid; TIMP, tissue inhibitor of metalloproteinase; EIA, enzyme immuonassay; ROC, receiver operating characteristics; AUC, area under curve; GSTA, glutathione S transferase A1. † Current address: Visiting Professor of Pediatrics and Nutrition, Division of Gastroenterology, Dept of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA.
1. Introduction Liver disease is a serious complication for patients with cystic fibrosis (CF), where the principal cause of morbidity and mortality is fibrosis leading to cirrhosis. The prevalence of liver disease at autopsy is 72% [1], yet clinically significant fibrosis during life is difficult to determine as no adequate diagnostic test is available. Significant morbidity from CF-associated liver disease (CFLD) occurs in 13–17% of children with CF and cirrhosis is detected in 10% [2]. Traditional measures of liver function do not correlate with severity of liver fibrosis [3,4] and diagnosis is frequently not made until complications such as portal hypertension are
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.06.032
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
established. Liver biopsy remains the standard for diagnosis, however, it is invasive and potentially confounded by sampling error as CFLD which, when established is a focal biliary cirrhosis [5], progressing to multilobular cirrhosis [6]. Hence the challenge exists to develop a sensitive, reliable non-invasive assay to identify patients with CF who are at risk of developing significant liver disease and to monitor progression or response to therapy [7]. The common pathway for liver diseases leading to cirrhosis, including CFLD [8], is via fibrogenesis, due to an imbalance between the synthesis and degradation of extracellular matrix by hepatic stellate cells (HSC) resulting in increased matrix deposition. Evaluation of hepatic fibrosis, by measuring serum levels of components of matrix degradation is well described in adult human liver diseases. Elevated serum (i) procollagen III polypeptide (PIIIP), collagen type-IV (CL-IV), prolyl hydroxylase (PH) [9], and matrix metalloproteinase-1 (MMP-1) [10], were seen in cirrhosis of various causes, (ii) laminin and CL-IV in alcoholic hepatitis [11], and (iii) hyaluronic acid (HA) [12], tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 [13], in chronic hepatitis C. Serum CL-IV, MMP-2 and TIMP-1 (but not laminin, MMP-1 or MMP-3) were elevated in hereditary haemochromatosis [14,15], and only CL-IV and MMP-2 correlated with severity of hepatic fibrosis [15]. These varying results demonstrate the disease-specific differences in the utility of serum markers of hepatic fibrosis. Serum markers of matrix remodelling can be potentially confounded by growth, as shown by increased bone and kidney expression of MMPs and TIMP-1 in the developing child [16,17]. However, serum PIIIP, CL-IV and laminin were elevated above that of age-matched controls in Indian Childhood Cirrhosis [18]. Further potential confounders include the multi-systemic nature of CF and inflammation. For example, MMPs and TIMP-1 are involved in bronchial tissue remodelling in asthma [19]. Nevertheless, the clinical imperative for such a disease marker in CFLD requires that candidate serum markers be evaluated in a controlled clinical setting. Previous studies have shown an elevation in collagen type-VI [20], PIIIP, PH [21] and HA [22] in children with CFLD. However, no study has specifically investigated these serum markers in a well-characterised cohort of children with CFLD, compared to age- and sex-matched controls and no correlations have been made with the degree of hepatic fibrosis assessed by liver biopsy. In this study we measured serum concentrations of CL-IV, PH, HA, MMP-2 and TIMP-1 in patients with CFLD versus age- and sexmatched patients with CF but no liver disease (CFnoLD) and non-CF controls. We evaluated the potential of these serum markers to predict the degree of fibrosis in CFLD, particularly to detect early disease, which might then be subject to longitudinal study as markers of progression of CFLD.
577
2. Methods 2.1. Study subjects Informed consent was obtained from parents of all patients. The protocol was approved by the Ethics Committees of The Queensland Institute of Medical Research and The Royal Children’s Hospital, Brisbane. CFLD was diagnosed in 36 children (mean age 11.2G0.7 years, 17 males, range 1.3– 18.8 years) showing at least two of the following: (i) hepatomegalyG splenomegaly; (ii) persistent elevation of serum alanine aminotransferase (ALT O1 ! Upper Limit Normal) longer than 6 months; (iii) ultrasound scan with abnormal echogenicity or nodular edge suggestive of cirrhosis. Thirty children were designated as CFnoLD, with absence of liver disease by clinical, biochemical and ultrasonographic modalities (mean age 10.4G 0.9 years, 21 males, range 0.3–16.5 years). Thirty-nine healthy children (mean age 9.2G0.5 years, 24 males, range 4.6–15.1 years) attending the hospital for minor plastic procedures served as controls. Blood was collected from all children and serum stored at K70 8C until assayed. Liver biopsy was performed on 31 CFLD patients under general anaesthetic.
2.2. Serum assays Serum CL-IV, MMP-2, TIMP-1, PH (Fuji Chemicals, Toyama, Japan) and HA (Chugai Diagnostics, Tokyo, Japan) were measured using one-step sandwich enzyme immunoassays (EIA), according to the manufacturer’s instructions.
2.3. Histopathological assessment of cystic fibrosis liver disease liver biopsies Liver tissue from 31 children with CFLD was fixed in 10% buffered formalin and paraffin-embedded. At least 10 levels of each biopsy were evaluated following staining with haematoxylin/eosin, haematoxylin/van Gieson, orcin, periodic Schiff-diastase, Perls’ and a silver-impregnated method for reticulin. Biopsies had a minimum of five portal tracts (range 5–13). The severity of fibrosis was assessed with the pathologist blinded to clinical and serum characteristics, using the Scheuer scoring method [23]. Sampling was improved by performing dual-pass biopsies. If fibrosis scores between the two biopsies were discordant, the highest (worst) score was correlated to serum markers. Inflammation was evaluated [23] and the presence of steatosis was noted but not quantified.
2.4. Statistical analysis Statistical analysis was performed using SPSS version 11.5.0 (SPSS Inc., IL, USA). Comparisons between the three patient groups are expressed as meanGstandard error of the mean (SEM). Normally distributed data were analysed using one-way ANOVA and unpaired Student’s t-test and where data was not normally distributed, the Kruskal–Wallis one-way ANOVA and Mann–Whitney U-test were used, with P!0.05. Correlations between serum markers and (i) the stage of histological fibrosis in CFLD subjects were determined using Spearman rank correlation coefficient for discontinuous variables, (ii) age were determined using Pearson’s correlation for continuous variables. Receiver-operating characteristics (ROC) analysis and logistic regression were carried out to verify the ability of these markers, both individually and in combination, to distinguish between patient groups and to identify early hepatic fibrogenesis in CFLD patients.
3. Results 3.1. Clinical characteristics of cystic fibrosis liver disease subjects The clinical characteristics of CFLD patients are found in Table 1. There was clinical evidence of hepatomegalyG splenomegaly in 25 (69%); definite portal hypertension in
578
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
Table 1 Clinical and histopathological characteristics of patients with CFLD No.
Sex
Age (yrs)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
F F F M M M F F M F F M M M M F M M M F M F F F F F F F M M F F F M M M
15.6 16.4 9.9 15.6 10.3 8.4 9.2 14.8 10 11.4 10.9 1.3 18.7 9.9 13.4 8.9 15.4 9.1 10.0 8.7 2.7 17.0 15.0 10.5 11.3 6.5 7.9 8.9 8.0 10.8 15.6 4.7 7.8 11.7 0.3 12
MI
IDDM
Clinical HSM
PHT
Infl (0–3)
Scheuer fibrosis score (0–4)
CF genotype
Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Yes Yes
1 1 1 1 1 2 2 NA 1 2 1 1 NA 1 1 2 2 NA 2 1 1 1 1 NA 2 0 0 0 NA 0 0 NA NA NA NA NA
4a 4a 3a 3a 3a 3a 3a 3 3 2a 2a 2 2a 2 2 2a 2a 1a 1 1a 1 1a 1a 1 1a 0 0 0 0 0a 0a NA NA NA NA NA
? DF/DF DF/DF DF/DF ? DF/DF DF/DF DF/DF DF/K DF/K ? DF/K ? DF/DF DF/DF DF/K ? DF/DF ? ? DF/DF ? DF/DF DF/K ? DF/DF ? DF/DF DF/DF ? DF/DF ?
Yes Yes Yes
Yes
Yes
Yes Yes Yes Yes Yes
Yes Yes
Yes Yes Yes Yes Yes
Yes Yes Yes Yes
Yes
Yes
Yes Yes
Yes Yes Yes
Yes
Yes Yes Yes
Yes
Yes Yes Yes Yes Yes Yes Yes
Yes Yes
DF/DF DF/DF DF/DF
MI, meconium ileus; IDDM, insulin-dependent diabetes mellitus; PH, portal hypertension; HSM, hepatomegalyGsplenomegaly; Infl, inflammatory score; NA, not available; DF, DF508. a Denotes the presence of steatosis.
13 (36%); a history of meconium ileus at birth in 11 (31%) and seven had insulin-dependent diabetes. The histopathological assessment of CFLD liver biopsies revealed fibrosis scores of zero in 6, one in 8, two in 9, three in 6 and four in 2 children. Steatosis was seen in 19 of 31 biopsies and as the only histopathological abnormality in two patients with grade 0 fibrosis. 3.2. Serum assays Serum TIMP-1 was significantly increased in CFLD patients (315G17 ng/ml) compared to both CFnoLD (249G 15 ng/ml) and control subjects (216G12 ng/ml) (ANOVA P!0.001) (Fig. 1A). In the CFLD patients there was a negative correlation between serum TIMP-1 levels and the stage of histological fibrosis with a trend towards statistical significance (rZK0.34; PZ0.06) (Fig. 1B). TIMP-1 levels
were significantly higher in CFLD patients without histological evidence of hepatic fibrosis, i.e. stage 0 (423G58 ng/ml), compared to both CFnoLD (PZ0.04) and controls (PZ0.02). Similarly, serum PH was significantly increased in CFLD patients (68G3 ng/ml) compared to both CFnoLD (54G 3 ng/ml) and control subjects (57G2 ng/ml), (ANOVA PZ 0.008) (Fig. 2A). There was a significant negative correlation between serum PH and the stage of histological fibrosis (rZK0.48; PZ0.008) (Fig. 2B). PH levels were significantly higher in CFLD patients without significant histological evidence of hepatic fibrosis, both in stage 0 (69.6G 4.2 ng/ml) and stage 1 fibrosis (81.8G8.9 ng/ml), compared to both CFnoLD (P!0.02) and controls (P!0.03). Serum CL-IV was significantly increased in CFLD patients (291G23 ng/ml) compared to CFnoLD (175G 15 ng/ml) and control subjects (173G9 ng/ml), (ANOVA
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
Fig. 1. (A) Serum TIMP-1 levels in patients with CFLD, CFnoLD and in control subjects. Serum TIMP-1 was significantly higher in CFLD patients compared to both CFnoLD patients and control subjects (ANOVA P!0.001). (B) Linear regression analysis of the correlation between serum TIMP-1 levels and the histological stage of hepatic fibrosis in patients with CFLD (rZK0.34, PZ0.06).
P!0.001) (Fig. 3). While this result suggests a potential liver-specific role for CL-IV in CF, there was no correlation between serum CL-IV and the stage of histological fibrosis in CFLD (results not shown). Serum HA was significantly increased in CFLD patients (23.7G2.3 ng/ml) compared to control subjects (18.0G 1.5 ng/ml, P!0.05), but not when compared to CFnoLD patients (19.5G1.6 ng/ml) (Fig. 4). There was no significant correlation between serum HA and the stage of histological fibrosis (results not shown). There was no significant difference in serum MMP-2 levels between any of the groups studied (CFLD, 1081G 46 ng/ml; CFnoLD, 985G51 ng/ml; controls, 1021G 47 ng/ml) and no correlation between serum MMP-2 and the stage of histological fibrosis (results not shown). There was no significant difference in either serum TIMP-1, CL-IV, PH or HA between control subjects and CFnoLD patients.
579
Fig. 2. (A) Serum PH levels in patients with CFLD, CFnoLD and in control subjects. Serum PH was significantly higher in CFLD patients compared to both CFnoLD patients and control subjects (ANOVA PZ0.008). (B) Linear regression analysis of the correlation between serum PH levels and the histological stage of hepatic fibrosis in patients with CFLD (rZK0.48, PZ0.008).
either CFnoLD or controls (Table 2), suggesting these markers are independent of growth. In the age-matched CFLD group there was a negative correlation between age and serum CLIV (rZK0.436, PZ0.009), TIMP-1 (rZK0.370, PZ0.027) and MMP-2 (rZK0.372, PZ 0.031). There was no correlation between age and either fibrosis stage (rZ0.2645, PZ0.157) or inflammation score (results not shown), nor were there correlations between any
3.3. Correlation between age and serum markers, fibrosis grade and inflammation score There was no correlation between any of the five serum markers and age of CFLD, CFnoLD and controls, grouped together (results not shown). When analysed separately, no correlation was observed between serum markers and age in
Fig. 3. Serum CL-IV levels in patients with CFLD and CFnoLD and in control subjects. Serum CL-IV was significantly higher in CFLD patients compared to CFnoLD patients and control subjects (ANOVA P!0.001).
580
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583 Table 3 Diagnostic value of individual serum markers for differentiating patients with CFLD from CFnoLD patients and controls Serum assay
CLIV TIMP-1 PH HA MMP-2
Fig. 4. Serum HA levels in patients with CFLD, CFnoLD and in control subjects. Serum HA was significantly higher in CFLD patients compared to control subjects (PZ0.05), but not when compared to patients with CFnoLD.
of the five serum markers and inflammation score (results not shown). 3.4. Diagnostic performance of serum assays 3.4.1. Discriminating liver disease in cystic fibrosis Table 3 shows the results of ROC curve analysis of individual assays, in order of ability to differentiate CFLD patients from CFnoLD patients and controls. Based on the area under the curve (AUC), CLIV, TIMP-1 and PH demonstrated the highest accuracy. The cut-off values with highest sensitivity and specificity for diagnosing CFLD were 215 ng/ml serum CLIV, 233 ng/ml serum TIMP-1 and 66 ng/ml serum PH. HA and MMP-2 had smaller AUC and were less informative. Table 4 shows the results of logistic regression analysis, used to combine individual assays to construct a single score for diagnosing CFLD. The combination of CLIV, TIMP-1 and PH, based on the following equation, gave a score with the highest overall accuracy: expf7:068K0:005XTIMP1K0:0110XCLIVK0:0469XPH g PZ 1Cexpf7:068K0:005XTIMP1K0:0110XCLIVK0:0469XPH g P is the probability of inclusion in the liver disease group. A score greater the 0.5 denotes a CFLD patient and a score less than 0.5, no liver disease. Fig. 5 shows the ROC curve for the three most useful individual assays, CLIV, TIMP-1 and Table 2 Correlation (linear regression) of serum markers with age Serum assay
CFLD patients r (P)
CFnoLD patients r (P)
Control subjects r (P)
TIMP-1 CLIV MMP-2 PH HA
K0.370 (0.027) K0.436 (0.009) K0.372 (0.031) K0.053 (0.777) K0.157 (0.389)
K0.240 (0.265) K0.149 (0.488) 0.102 (0.627) K0.053 (0.810) 0.082 (0.711)
K0.09 (0.642) 0.044 (0.822) 0.197 (0.256) 0.273 (0.196) 0.032 (0.859)
ROC AUC
P
% sensitivity
% specificity
Cut-off (ng/ml)
0.785 0.765 0.688 0.611 0.598
!0.0001 !0.0001 0.0011 0.0385 0.0453
69.4 83.3 45.2 46.9 62.9
81.7 61.5 88.2 73.0 59.7
215 232.8 66 20.8 998
PH as well as the curve (based on logistic regression scores) for the combination of the three assays. 3.4.2. Discriminating early hepatic fibrogenesis in cystic fibrosis Fig. 6(A) shows the ROC curve for serum PH (AUC 0.814, P!0.0001), which was useful in distinguishing CFLD patients with early fibrogenesis (stages 0 and 1) from those with extensive fibrosis (stages 2–4), with a cut-off level of 59 ng/ml (60% sensitive and 91.7% specific). Serum TIMP-1 showed a trend towards statistical significance in differentiating CFLD patients with no fibrosis (stage 0) from those with fibrosis (AUC 0.725, PZ0.06), with a cut-off at 300 ng/ml (64% sensitive and 83.3% specific) (results not shown). The fit of the model and thus the diagnostic accuracy of assessing CFLD patients with ‘no fibrosis’ vs. ‘fibrosis’ was improved (AUC 0.852, P!0.02) by constructing the ROC curve combining PH and TIMP-1 with logistic regression (Fig. 6B), based on the following equation: PZ
expf8:917 K 0:021XTIMP1 K 0:005XPH g 1 C expf8:917 K 0:021XTIMP1 K 0:005XPH g
P is the probability of inclusion in the ‘fibrosis’ group. A score greater the 0.5 denoting fibrosis in a patient with CFLD (stages 1–4) and a score less than 0.5, indicating no fibrosis (stage 0). Table 4 Diagnostic value of combined serum markers for differentiating patients with CFLD from CFnoLD patients and controls Serum Assay
CLIV and PH CLIV and TIMP-1 TIMP-1 and PH CLIV, TIMP-1 and PH CLIV, TIMP-1 and HA CLIV, TIMP-1, HA and PH all five assays a
ROC AUCa
Logistic regression P
% Sensitivity
% Specificity
% total
0.829 0.807 0.771 0.831
!0.0001 !0.0001 !0.0001 !0.0001
89.8 89.83 86.27 87.76
67.74 66.67 51.61 70.97
81.25 81.05 73.17 81.25
0.805
!0.0001 92.86
65.62
82.95
0.838
!0.0001 89.9
66.67
81.01
0.847
!0.0001 85.71
66.67
78.48
ROC AUC based on logistic regression scores.
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
581
Fig. 5. ROC curves for serum concentrations of CL-IV, TIMP-1 and PH, individually and in combination, for discriminating CFLD patients from those CF patients without liver disease and Controls. The AUC are 0.785 (P!0.0001), 0.765 (P!0.0001), 0.688 (PZ0.0011) and 0.831 (P!0.0001) for CL-IV, TIMP-1, PH and all three combined, respectively.
The logistic model fitted to differentiate ‘early fibrogenesis’ (stages 0 and 1) vs. ‘extensive fibrosis’ (stages 2–4), that included TIMP-1 with PH yielded a ROC AUC of 0.833 (P!0.01) (results not shown). However, this modelling did not significantly improve the diagnostic accuracy when compared to serum PH results alone.
4. Discussion This study evaluated the utility of a number of serum markers of hepatic fibrosis, individually and in combination, in a well characterised cohort of patients with CFLD, where liver biopsies were available for scoring of fibrosis, versus age- and sex-matched patients with CFnoLD and non-CF controls. We demonstrated that serum TIMP-1, PH and CL-IV levels were significantly higher in patients with CFLD compared to CFnoLD and controls, suggesting relative specificity for liver injury in CF. Serum HA was significantly increased in CFLD compared to controls, but not when compared to CFnoLD, indicating the extra-hepatic confounding influences in patients with CF. In addition, serum TIMP-1 and PH were significantly higher in CFLD subjects with minimal or no histological evidence of fibrosis suggesting a potential role for these markers in the early detection of liver injury. When used in combination, serum levels of CL-IV, TIMP-1 and PH demonstrated improved diagnostic accuracy in differentiating CFLD from both CFnoLD and controls, compared to using individual assays. A combination of serum TIMP-1 and PH showed greater diagnostic
Fig. 6. (A) ROC curve for the serum concentration of PH in discriminating patients with early fibrosis (stages 0 and 1) from those with extensive fibrosis (stages 2–4). The area under the curve is 0.814 (P!0.0001). (B). ROC curve combining serum PH and TIMP-1 with logistic regression, for identifying CFLD patients with no fibrosis (stage 0) vs. those with any fibrosis (stages 1–4). The ROC AUC is 0.852 (P!0.02).
accuracy in identifying CFLD patients with no fibrosis (stage 0) from those with fibrosis (stages 1–4). While the pathogenesis of liver disease in CF remains to be determined, we have demonstrated that activated HSC are the cellular source of increased procollagen a1(I) mRNA expression in CFLD and thus are responsible for the hepatic fibrosis and focal biliary cirrhosis seen in CF [8]. Measuring the products of HSC activity less invasively in serum is an attractive proposition for monitoring fibrosis provided the confounding influences of growth and non-hepatic inflammation are minimal. Several studies have attempted to address the utility of serum markers of hepatic fibrosis in CF. Serum collagen VI was studied in 30 children with liver biopsy-verified CFLD,
582
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
133 children with CF and no detectable liver disease (by clinical and conventional blood tests only) and controls [20]. Levels were independent of age, all patients with fibrosis had elevated levels and there was no overlap with controls. However, no stratification was undertaken with severity of fibrosis. Two studies have suggested that human glutathione S transferase A1 (GSTA) was superior to conventional transaminases and comparable to ultrasonography in detecting liver disease, but its utility for detecting hepatic fibrosis was not determined as no biopsies were undertaken [24,25]. Serum PIIIP and PH were studied in 44 children with CF, 26 with suggestion of liver disease by clinical and ultrasound abnormalities [21]. Only four underwent liver biopsy and no comparison of serum markers was made between CFLD and CFnoLD. As a group, children with CF had higher serum PH but not PIIIP verses controls [21]. We related serum markers to histological severity of fibrosis, rather than liver dysfunction, in keeping with other publications. Several patients with only moderate to advanced histological fibrosis had evidence of portal hypertension, a clinical indicator of more advanced liver dysfunction. While our dual-pass biopsy sampling may underestimate the severity of fibrosis in some children, portal hypertension is not synonymous with cirrhosis. Moreover, it seemingly develops earlier in the natural history of CFLD than in other fibrosing liver diseases. The major clinical importance of serum markers remains in the detection and monitoring of hepatic fibrosis before advanced dysfunction becomes evident. TIMP-1 inhibits the activity of MMPs, which degrade extracellular matrix during tissue remodelling associated with active fibrogenesis [26]. TIMP-1 is produced by activated HSC and elevated serum TIMP-I reflects active fibrogenesis in HCV [13], haemochromatosis [14] and alcoholic liver disease [27]. TIMP-1 is active in various organs and disease states, including the failing heart, colitis and asthma [19]. We found serum TIMP-1 significantly elevated in children with CFLD compared to those without liver disease. Using ROC analysis we also showed that serum TIMP-1 might be useful as a diagnostic marker to distinguish between these groups. Early CFLD is associated with marked elevations of TIMP-1, which decreases with progression of fibrosis. This result is similar to previous studies in HCV [13] and haemochromatosis [14] and points to a role for TIMP-1 in remodelling extracellular matrix in early hepatic fibrogenesis in CF. Combining serum TIMP-1 with PH markedly improved the diagnostic accuracy in identifying CFLD patients with fibrosis vs. no fibrosis. PH is an enzyme required for collagen biosynthesis and is produced in the liver by hepatocytes, HSC and endothelial cells. Leonardi and colleagues showed that serum PH was elevated in children with CF vs. non-CF controls, with a non-significant increase in PH levels in four children suspected of having liver disease [21]. Our results show serum PH is elevated in CFLD vs. both CFnoLD
and controls and negatively correlated with hepatic fibrosis scores. This was verified with ROC analysis, demonstrating that serum PH showed high predictive value in distinguishing between patients with early, active hepatic fibrogenesis versus late stage fibrosis, as was previously reported in early alcoholic cirrhosis in adults [9]. This study is the first to show that serum CL-IV is elevated in children with CFLD vs. CFnoLD and controls and thus may identify CF patients with liver disease. Furthermore, when serum levels of CL-IV were used in combination with TIMP-1 and PH, the diagnostic accuracy in identifying patients with CFLD vs. CFnoLD and controls was improved. CL-IV is a major component of basement membrane. Unlike TIMP-1 and PH, serum CL-IV levels were elevated even in more advanced fibrosis. This is not surprising as with fibrosis progression, more CL-IV is degraded by MMPs and the EIA used in this study measures degradation products of CL-IV. Others have also shown sustained elevation in serum CL-IV in chronic liver disease [9,11]. However, contrary to reports in other chronic adult liver diseases [15], CL-IV is not a quantitative marker for progression of fibrosis in CFLD. While we demonstrated that HA was elevated in CFLD vs. controls, there was no significant difference between CFLD and CFnoLD, suggesting HA is less specific for CF liver injury. Elevated serum HA was previously seen in CFLD vs. both CFnoLD and controls [22]. However, as liver biopsy was not performed and no stratification with fibrosis undertaken, CFLD patients may have had more advanced liver disease, which could explain why this study showed a significant difference between CFLD vs. CFnoLD. Others have shown that serum HA is elevated in adult alcoholic liver disease, chronic hepatitis C viral infection and primary biliary cirrhosis [12,28,29]. In these diseases elevated HA levels correlated with the degree of fibrosis. In our study, serum HA did not correlate with histological fibrosis and thus, it is not a useful marker in CFLD. Serum MMP-2 was uninformative in distinguishing CF patients with or without liver disease, from controls. Studies in adults have provided conflicting results on the utility of MMP-2 as a marker of hepatic fibrosis. We previously demonstrated a correlation between serum MMP-2 and fibrosis in haemochromatosis [14]. However, Lichtinghagen and colleagues showed that serum MMP-2 did not correlate with fibrosis in patients with chronic active hepatitis and hepatitis C-induced cirrhosis [30]. These varying data clearly demonstrate the disease-specific utility of individual serum markers. In summary, we have shown that serum TIMP-1, PH and CL-IV are increased in patients with CFLD compared to CFnoLD and control subjects. Serum TIMP-1 and PH levels are elevated in early hepatic fibrogenesis and return to control levels with progression to advanced fibrosis. Serum TIMP-1 and PH may be markers of early events associated with CFLD, prior to significant scar tissue deposition, and thus may be useful in the early detection of liver disease in
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
CF, particularly when used in combination. We suggest these serum markers require assessment in longitudinal studies of CFLD to determine their utility for predicting significant liver disease and as markers of disease progression.
Acknowledgements This project was funded by a Major Research Project Grant (#913-005 to PJL, JLS, RWS, GAR) and a Seeding Grant (#730 to PJL, JLS, RWS, GAR) from the Royal Children’s Hospital Foundation, Brisbane and by a grant from the National Health and Medical Research Council of Australia (Grant No. 290220 to GAR and PJL).
References [1] Vawter GF, Shwachman H. Cystic fibrosis in adults: an autopsy study. Pathol Annu 1979;14:357–382. [2] Sokol RJ, Durie PR. Recommendations for management of liver and biliary tract disease in cystic fibrosis. Cystic Fibrosis Foundation Hepatobiliary Disease Consensus Group. J Pediatr Gastroenterol Nutr 1999;28:S1–S13. [3] Tanner MS. Liver and biliary problems in cystic fibrosis. J R Soc Med 1992;85:20–24. [4] Williams SG, Westaby D, Tanner MS, Mowat AP. Liver and biliary problems in cystic fibrosis. Br Med Bull 1992;48:877–892. [5] Weber AM, Lenaerts C, Smith L, Roy CC. Screening and diagnosis of cystic fibrosis-related liver disease. Pediatr Pulmonol 1989;Suppl 4: 40–41. [6] Hultcrantz R, Mengarelli S, Strandvik B. Morphological findings in the liver of children with cystic fibrosis: a light and electron microscopical study. Hepatology 1986;6:881–889. [7] Lowe ME, Ameen N, Freedman S, Mullberg A, Werlin SL. Research agenda for pediatric gastroenterology, hepatology and nutrition: cystic fibrosis and pancreatic diseases. Report of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition for the Children’s Digestive Health and Nutrition Foundation. J Pediatr Gastroenterol Nutr 2002;35:S258–S262. [8] Lewindon PJ, Pereira TN, Hoskins AC, Bridle KR, Williamson RM, Shepherd RW, et al. The role of hepatic stellate cells and transforming growth factor-beta(1) in cystic fibrosis liver disease. Am J Pathol 2002;160:1705–1715. [9] Fabris C, Falleti E, Federico E, Toniutto P, Pirisi M. A comparison of four serum markers of fibrosis in the diagnosis of cirrhosis. Ann Clin Biochem 1997;34:151–155. [10] Ueno T, Tamaki S, Sugawara H, Inuzuka S, Torimura T, Sata M, et al. Significance of serum tissue inhibitor of metalloproteinases-1 in various liver diseases. J Hepatol 1996;24:177–184. [11] Castera L, Hartmann DJ, Chapel F, Guettier C, Mall F, Lons T, et al. Serum laminin and type IV collagen are accurate markers of histologically severe alcoholic hepatitis in patients with cirrhosis. J Hepatol 2000;32:412–418. [12] Guechot J, Laudat A, Loria A, Serfaty L, Poupon R, Giboudeau J. Diagnostic accuracy of hyaluronan and type III procollagen amino-terminal peptide serum assays as markers of liver fibrosis in chronic viral hepatitis C evaluated by ROC curve analysis. Clin Chem 1996;42:558–563.
583
[13] Walsh KM, Timms P, Campbell S, MacSween RN, Morris AJ. Plasma levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2) as noninvasive markers of liver disease in chronic hepatitis C: comparison using ROC analysis. Dig Dis Sci 1999;44:624–630. [14] George DK, Ramm GA, Powell LW, Fletcher LM, Walker NI, Cowley LL, et al. Evidence for altered hepatic matrix degradation in genetic haemochromatosis. Gut 1998;42:715–720. [15] George DK, Ramm GA, Walker NI, Powell LW, Crawford DH. Elevated serum type IV collagen: a sensitive indicator of the presence of cirrhosis in haemochromatosis. J Hepatol 1999;31:47–52. [16] Thrailkill KM, Kumar S, Rosenberg CK, Auten KJ, Fowlkes JL. Characterization of matrix metalloproteinases in human urine: alterations during adolescence. Pediatr Nephrol 1999;13:223–229. [17] Bord S, Horner A, Beeton CA, Hembry RM, Compston JE. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) distribution in normal and pathological human bone. Bone 1999;24:229–235. [18] Trivedi P, Risteli J, Risteli L, Tanner MS, Bhave S, Pandit AN, et al. Serum type III procollagen and basement membrane proteins as noninvasive markers of hepatic pathology in Indian childhood cirrhosis. Hepatology 1987;7:1249–1253. [19] Hoshino M, Takahashi M, Takai Y, Sim J. Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma. J Allergy Clin Immunol 1999;104:356–363. [20] Gerling B, Becker M, Staab D, Schuppan D. Prediction of liver fibrosis according to serum collagen VI level in children with cystic fibrosis. N Engl J Med 1997;336:1611–1612. [21] Leonardi S, Giambusso F, Sciuto C, Castigliones S, Castiglione N, La Rosa M. Are serum type III procollagen and prolyl hydroxylase useful as noninvasive markers of liver disease in patients with cystic fibrosis? J Pediatr Gastroenterol Nutr 1998;27:603–605. [22] Wyatt HA, Dhawan A, Cheeseman P, Mieli-Vergani G, Price JF. Serum hyaluronic acid concentrations are increased in cystic fibrosis patients with liver disease. Arch Dis Child 2002;86:190–193. [23] Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991;13:372–374. [24] Hung JC, Howie AF, Beckett GJ, Sood M, Hambleton G, Super M. The use of human glutathione S-transferase A1 in the detection of cystic fibrosis liver disease. J Paediatr Child Health 1998;34:335–338. [25] Sidlova K, Skalicka V, Kotaska K, Pechova M, Chada M, Barlosova J, et al. Serum alpha-glutathione S-transferase as a sensitive marker of hepatocellular damage in patients with cystic fibrosis. Physiol Res 2003;52:361–365. [26] Iredale JP. Tissue inhibitors of metalloproteinases in liver fibrosis. Int J Biochem Cell Biol 1997;29:43–54. [27] Li J, Rosman AS, Leo MA, Nagsˇi Y, Lieber CS. Tissue inhibitor of metalloproteinase is increased in the serum of precirrhotic and cirrhotic alcoholic patients and can serve as a marker of fibrosis. Hepatology 1994;19:1418–1423. [28] Pares A, Deulofeu R, Gimenez A, Caballeria L, Bruguera M, Caballeria J, et al. Serum hyaluronate reflects hepatic fibrogenesis in alcoholic liver disease and is useful as a marker of fibrosis. Hepatology 1996;24:1399–1403. [29] Nyberg A, Engstrom-Laurent A, Loof L. Serum hyaluronate in primary biliary cirrhosis—a biochemical marker for progressive liver damage. Hepatology 1988;8:142–146. [30] Lichtinghagen R, Huegel O, Seifert T, Haberkom CI, Michels D, Flemming P, et al. Expression of matrix metalloproteinase-2 and -9 and their inhibitors in peripheral blood cells of patients with chronic hepatitis C. Clin Chem 2000;46:183–192.
Serum markers of hepatic fibrogenesis in cystic fibrosis liver disease Tamara N. Pereira1, Peter J. Lewindon1,4, Jeffery L. Smith1,5, Therese L. Murphy2, Douglas J. Lincoln3, Ross W. Shepherd4,†, Grant A. Ramm1,* 1
The Hepatic Fibrosis Group, The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia 2 Iron Metabolism Group, The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia 3 Cancer and Population Studies Group, The Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Brisbane, Qld 4029, Australia 4 Department of Gastroenterology, Royal Children’s Hospital, Brisbane 4029, Australia 5 Department of Biochemistry and Molecular Biology, The University of Queensland, Brisbane 4029, Australia
Background/Aims: Hepatic fibrosis contributes to adverse outcome in cystic fibrosis (CF). Early detection of CF liver disease (CFLD) may identify patients at risk of significant complications. To evaluate the utility of serum markers to detect hepatic fibrosis in children with CFLD vs. CF patients without liver disease (CFnoLD) and controls. Methods: Sera from 36 CFLD, 30 CFnoLD and 39 controls were assessed for tissue inhibitor of matrix metalloproteinase (MMP) (TIMP)-1, collagen (CL)-IV, MMP-2, hyaluronic acid (HA) and prolyl hydroxylase (PH) by enzyme immunoassay and were correlated with hepatic fibrosis score in CFLD. Results: TIMP-1, PH and CL-IV were increased in CFLD vs. CFnoLD and controls. Fibrosis score was negatively correlated with TIMP-1 (rZK0.34, PZ0.06) and PH (rZK0.48, PZ0.008). Receiver-operating characteristics analysis showed CL-IV (AUC 0.785, P!0.0001) and TIMP-1 (AUC 0.765, P!0.0001) differentiated CFLD from CFnoLD and controls, while PH (AUC 0.814, P!0.0001) predicted early fibrogenesis. Diagnostic accuracy improved using logistic regression combining (i) CL-IV, TIMP-1, PH to identify CFLD (AUC 0.831, P!0.0001) and (ii) TIMP-1, PH to identify CFLD patients with no fibrosis (AUC 0.852, P!0.02). Conclusions: Elevated TIMP-1, CL-IV, PH may be indicators of hepatic fibrogenesis in CF. Increased TIMP-1, PH may be early markers of CFLD. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis liver disease; Serum markers of hepatic fibrosis; TIMP-1; Prolyl hydroxylase; Collagen type IV
Received 28 January 2004; received in revised form 21 May 2004; accepted 17 June 2004; available online 23 July 2004 * Corresponding author. Tel.: C61-7-3362-0177; fax: C61-7-33620191. E-mail address: [email protected] (G.A. Ramm). Abbreviations: CF, cystic fibrosis; CFLD, cystic fibrosis liver disease; CFnoLD, cystic fibrosis no liver disease; HSC, hepatic stellate cell; PIIIP, procollagen III polypeptide; CL-IV, collagen type IV; PH, prolyl hydroxylase; MMP, matrix metalloproteinase; HA, hyaluronic acid; TIMP, tissue inhibitor of metalloproteinase; EIA, enzyme immuonassay; ROC, receiver operating characteristics; AUC, area under curve; GSTA, glutathione S transferase A1. † Current address: Visiting Professor of Pediatrics and Nutrition, Division of Gastroenterology, Dept of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA.
1. Introduction Liver disease is a serious complication for patients with cystic fibrosis (CF), where the principal cause of morbidity and mortality is fibrosis leading to cirrhosis. The prevalence of liver disease at autopsy is 72% [1], yet clinically significant fibrosis during life is difficult to determine as no adequate diagnostic test is available. Significant morbidity from CF-associated liver disease (CFLD) occurs in 13–17% of children with CF and cirrhosis is detected in 10% [2]. Traditional measures of liver function do not correlate with severity of liver fibrosis [3,4] and diagnosis is frequently not made until complications such as portal hypertension are
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.06.032
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
established. Liver biopsy remains the standard for diagnosis, however, it is invasive and potentially confounded by sampling error as CFLD which, when established is a focal biliary cirrhosis [5], progressing to multilobular cirrhosis [6]. Hence the challenge exists to develop a sensitive, reliable non-invasive assay to identify patients with CF who are at risk of developing significant liver disease and to monitor progression or response to therapy [7]. The common pathway for liver diseases leading to cirrhosis, including CFLD [8], is via fibrogenesis, due to an imbalance between the synthesis and degradation of extracellular matrix by hepatic stellate cells (HSC) resulting in increased matrix deposition. Evaluation of hepatic fibrosis, by measuring serum levels of components of matrix degradation is well described in adult human liver diseases. Elevated serum (i) procollagen III polypeptide (PIIIP), collagen type-IV (CL-IV), prolyl hydroxylase (PH) [9], and matrix metalloproteinase-1 (MMP-1) [10], were seen in cirrhosis of various causes, (ii) laminin and CL-IV in alcoholic hepatitis [11], and (iii) hyaluronic acid (HA) [12], tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 [13], in chronic hepatitis C. Serum CL-IV, MMP-2 and TIMP-1 (but not laminin, MMP-1 or MMP-3) were elevated in hereditary haemochromatosis [14,15], and only CL-IV and MMP-2 correlated with severity of hepatic fibrosis [15]. These varying results demonstrate the disease-specific differences in the utility of serum markers of hepatic fibrosis. Serum markers of matrix remodelling can be potentially confounded by growth, as shown by increased bone and kidney expression of MMPs and TIMP-1 in the developing child [16,17]. However, serum PIIIP, CL-IV and laminin were elevated above that of age-matched controls in Indian Childhood Cirrhosis [18]. Further potential confounders include the multi-systemic nature of CF and inflammation. For example, MMPs and TIMP-1 are involved in bronchial tissue remodelling in asthma [19]. Nevertheless, the clinical imperative for such a disease marker in CFLD requires that candidate serum markers be evaluated in a controlled clinical setting. Previous studies have shown an elevation in collagen type-VI [20], PIIIP, PH [21] and HA [22] in children with CFLD. However, no study has specifically investigated these serum markers in a well-characterised cohort of children with CFLD, compared to age- and sex-matched controls and no correlations have been made with the degree of hepatic fibrosis assessed by liver biopsy. In this study we measured serum concentrations of CL-IV, PH, HA, MMP-2 and TIMP-1 in patients with CFLD versus age- and sexmatched patients with CF but no liver disease (CFnoLD) and non-CF controls. We evaluated the potential of these serum markers to predict the degree of fibrosis in CFLD, particularly to detect early disease, which might then be subject to longitudinal study as markers of progression of CFLD.
577
2. Methods 2.1. Study subjects Informed consent was obtained from parents of all patients. The protocol was approved by the Ethics Committees of The Queensland Institute of Medical Research and The Royal Children’s Hospital, Brisbane. CFLD was diagnosed in 36 children (mean age 11.2G0.7 years, 17 males, range 1.3– 18.8 years) showing at least two of the following: (i) hepatomegalyG splenomegaly; (ii) persistent elevation of serum alanine aminotransferase (ALT O1 ! Upper Limit Normal) longer than 6 months; (iii) ultrasound scan with abnormal echogenicity or nodular edge suggestive of cirrhosis. Thirty children were designated as CFnoLD, with absence of liver disease by clinical, biochemical and ultrasonographic modalities (mean age 10.4G 0.9 years, 21 males, range 0.3–16.5 years). Thirty-nine healthy children (mean age 9.2G0.5 years, 24 males, range 4.6–15.1 years) attending the hospital for minor plastic procedures served as controls. Blood was collected from all children and serum stored at K70 8C until assayed. Liver biopsy was performed on 31 CFLD patients under general anaesthetic.
2.2. Serum assays Serum CL-IV, MMP-2, TIMP-1, PH (Fuji Chemicals, Toyama, Japan) and HA (Chugai Diagnostics, Tokyo, Japan) were measured using one-step sandwich enzyme immunoassays (EIA), according to the manufacturer’s instructions.
2.3. Histopathological assessment of cystic fibrosis liver disease liver biopsies Liver tissue from 31 children with CFLD was fixed in 10% buffered formalin and paraffin-embedded. At least 10 levels of each biopsy were evaluated following staining with haematoxylin/eosin, haematoxylin/van Gieson, orcin, periodic Schiff-diastase, Perls’ and a silver-impregnated method for reticulin. Biopsies had a minimum of five portal tracts (range 5–13). The severity of fibrosis was assessed with the pathologist blinded to clinical and serum characteristics, using the Scheuer scoring method [23]. Sampling was improved by performing dual-pass biopsies. If fibrosis scores between the two biopsies were discordant, the highest (worst) score was correlated to serum markers. Inflammation was evaluated [23] and the presence of steatosis was noted but not quantified.
2.4. Statistical analysis Statistical analysis was performed using SPSS version 11.5.0 (SPSS Inc., IL, USA). Comparisons between the three patient groups are expressed as meanGstandard error of the mean (SEM). Normally distributed data were analysed using one-way ANOVA and unpaired Student’s t-test and where data was not normally distributed, the Kruskal–Wallis one-way ANOVA and Mann–Whitney U-test were used, with P!0.05. Correlations between serum markers and (i) the stage of histological fibrosis in CFLD subjects were determined using Spearman rank correlation coefficient for discontinuous variables, (ii) age were determined using Pearson’s correlation for continuous variables. Receiver-operating characteristics (ROC) analysis and logistic regression were carried out to verify the ability of these markers, both individually and in combination, to distinguish between patient groups and to identify early hepatic fibrogenesis in CFLD patients.
3. Results 3.1. Clinical characteristics of cystic fibrosis liver disease subjects The clinical characteristics of CFLD patients are found in Table 1. There was clinical evidence of hepatomegalyG splenomegaly in 25 (69%); definite portal hypertension in
578
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
Table 1 Clinical and histopathological characteristics of patients with CFLD No.
Sex
Age (yrs)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
F F F M M M F F M F F M M M M F M M M F M F F F F F F F M M F F F M M M
15.6 16.4 9.9 15.6 10.3 8.4 9.2 14.8 10 11.4 10.9 1.3 18.7 9.9 13.4 8.9 15.4 9.1 10.0 8.7 2.7 17.0 15.0 10.5 11.3 6.5 7.9 8.9 8.0 10.8 15.6 4.7 7.8 11.7 0.3 12
MI
IDDM
Clinical HSM
PHT
Infl (0–3)
Scheuer fibrosis score (0–4)
CF genotype
Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Yes Yes
1 1 1 1 1 2 2 NA 1 2 1 1 NA 1 1 2 2 NA 2 1 1 1 1 NA 2 0 0 0 NA 0 0 NA NA NA NA NA
4a 4a 3a 3a 3a 3a 3a 3 3 2a 2a 2 2a 2 2 2a 2a 1a 1 1a 1 1a 1a 1 1a 0 0 0 0 0a 0a NA NA NA NA NA
? DF/DF DF/DF DF/DF ? DF/DF DF/DF DF/DF DF/K DF/K ? DF/K ? DF/DF DF/DF DF/K ? DF/DF ? ? DF/DF ? DF/DF DF/K ? DF/DF ? DF/DF DF/DF ? DF/DF ?
Yes Yes Yes
Yes
Yes
Yes Yes Yes Yes Yes
Yes Yes
Yes Yes Yes Yes Yes
Yes Yes Yes Yes
Yes
Yes
Yes Yes
Yes Yes Yes
Yes
Yes Yes Yes
Yes
Yes Yes Yes Yes Yes Yes Yes
Yes Yes
DF/DF DF/DF DF/DF
MI, meconium ileus; IDDM, insulin-dependent diabetes mellitus; PH, portal hypertension; HSM, hepatomegalyGsplenomegaly; Infl, inflammatory score; NA, not available; DF, DF508. a Denotes the presence of steatosis.
13 (36%); a history of meconium ileus at birth in 11 (31%) and seven had insulin-dependent diabetes. The histopathological assessment of CFLD liver biopsies revealed fibrosis scores of zero in 6, one in 8, two in 9, three in 6 and four in 2 children. Steatosis was seen in 19 of 31 biopsies and as the only histopathological abnormality in two patients with grade 0 fibrosis. 3.2. Serum assays Serum TIMP-1 was significantly increased in CFLD patients (315G17 ng/ml) compared to both CFnoLD (249G 15 ng/ml) and control subjects (216G12 ng/ml) (ANOVA P!0.001) (Fig. 1A). In the CFLD patients there was a negative correlation between serum TIMP-1 levels and the stage of histological fibrosis with a trend towards statistical significance (rZK0.34; PZ0.06) (Fig. 1B). TIMP-1 levels
were significantly higher in CFLD patients without histological evidence of hepatic fibrosis, i.e. stage 0 (423G58 ng/ml), compared to both CFnoLD (PZ0.04) and controls (PZ0.02). Similarly, serum PH was significantly increased in CFLD patients (68G3 ng/ml) compared to both CFnoLD (54G 3 ng/ml) and control subjects (57G2 ng/ml), (ANOVA PZ 0.008) (Fig. 2A). There was a significant negative correlation between serum PH and the stage of histological fibrosis (rZK0.48; PZ0.008) (Fig. 2B). PH levels were significantly higher in CFLD patients without significant histological evidence of hepatic fibrosis, both in stage 0 (69.6G 4.2 ng/ml) and stage 1 fibrosis (81.8G8.9 ng/ml), compared to both CFnoLD (P!0.02) and controls (P!0.03). Serum CL-IV was significantly increased in CFLD patients (291G23 ng/ml) compared to CFnoLD (175G 15 ng/ml) and control subjects (173G9 ng/ml), (ANOVA
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
Fig. 1. (A) Serum TIMP-1 levels in patients with CFLD, CFnoLD and in control subjects. Serum TIMP-1 was significantly higher in CFLD patients compared to both CFnoLD patients and control subjects (ANOVA P!0.001). (B) Linear regression analysis of the correlation between serum TIMP-1 levels and the histological stage of hepatic fibrosis in patients with CFLD (rZK0.34, PZ0.06).
P!0.001) (Fig. 3). While this result suggests a potential liver-specific role for CL-IV in CF, there was no correlation between serum CL-IV and the stage of histological fibrosis in CFLD (results not shown). Serum HA was significantly increased in CFLD patients (23.7G2.3 ng/ml) compared to control subjects (18.0G 1.5 ng/ml, P!0.05), but not when compared to CFnoLD patients (19.5G1.6 ng/ml) (Fig. 4). There was no significant correlation between serum HA and the stage of histological fibrosis (results not shown). There was no significant difference in serum MMP-2 levels between any of the groups studied (CFLD, 1081G 46 ng/ml; CFnoLD, 985G51 ng/ml; controls, 1021G 47 ng/ml) and no correlation between serum MMP-2 and the stage of histological fibrosis (results not shown). There was no significant difference in either serum TIMP-1, CL-IV, PH or HA between control subjects and CFnoLD patients.
579
Fig. 2. (A) Serum PH levels in patients with CFLD, CFnoLD and in control subjects. Serum PH was significantly higher in CFLD patients compared to both CFnoLD patients and control subjects (ANOVA PZ0.008). (B) Linear regression analysis of the correlation between serum PH levels and the histological stage of hepatic fibrosis in patients with CFLD (rZK0.48, PZ0.008).
either CFnoLD or controls (Table 2), suggesting these markers are independent of growth. In the age-matched CFLD group there was a negative correlation between age and serum CLIV (rZK0.436, PZ0.009), TIMP-1 (rZK0.370, PZ0.027) and MMP-2 (rZK0.372, PZ 0.031). There was no correlation between age and either fibrosis stage (rZ0.2645, PZ0.157) or inflammation score (results not shown), nor were there correlations between any
3.3. Correlation between age and serum markers, fibrosis grade and inflammation score There was no correlation between any of the five serum markers and age of CFLD, CFnoLD and controls, grouped together (results not shown). When analysed separately, no correlation was observed between serum markers and age in
Fig. 3. Serum CL-IV levels in patients with CFLD and CFnoLD and in control subjects. Serum CL-IV was significantly higher in CFLD patients compared to CFnoLD patients and control subjects (ANOVA P!0.001).
580
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583 Table 3 Diagnostic value of individual serum markers for differentiating patients with CFLD from CFnoLD patients and controls Serum assay
CLIV TIMP-1 PH HA MMP-2
Fig. 4. Serum HA levels in patients with CFLD, CFnoLD and in control subjects. Serum HA was significantly higher in CFLD patients compared to control subjects (PZ0.05), but not when compared to patients with CFnoLD.
of the five serum markers and inflammation score (results not shown). 3.4. Diagnostic performance of serum assays 3.4.1. Discriminating liver disease in cystic fibrosis Table 3 shows the results of ROC curve analysis of individual assays, in order of ability to differentiate CFLD patients from CFnoLD patients and controls. Based on the area under the curve (AUC), CLIV, TIMP-1 and PH demonstrated the highest accuracy. The cut-off values with highest sensitivity and specificity for diagnosing CFLD were 215 ng/ml serum CLIV, 233 ng/ml serum TIMP-1 and 66 ng/ml serum PH. HA and MMP-2 had smaller AUC and were less informative. Table 4 shows the results of logistic regression analysis, used to combine individual assays to construct a single score for diagnosing CFLD. The combination of CLIV, TIMP-1 and PH, based on the following equation, gave a score with the highest overall accuracy: expf7:068K0:005XTIMP1K0:0110XCLIVK0:0469XPH g PZ 1Cexpf7:068K0:005XTIMP1K0:0110XCLIVK0:0469XPH g P is the probability of inclusion in the liver disease group. A score greater the 0.5 denotes a CFLD patient and a score less than 0.5, no liver disease. Fig. 5 shows the ROC curve for the three most useful individual assays, CLIV, TIMP-1 and Table 2 Correlation (linear regression) of serum markers with age Serum assay
CFLD patients r (P)
CFnoLD patients r (P)
Control subjects r (P)
TIMP-1 CLIV MMP-2 PH HA
K0.370 (0.027) K0.436 (0.009) K0.372 (0.031) K0.053 (0.777) K0.157 (0.389)
K0.240 (0.265) K0.149 (0.488) 0.102 (0.627) K0.053 (0.810) 0.082 (0.711)
K0.09 (0.642) 0.044 (0.822) 0.197 (0.256) 0.273 (0.196) 0.032 (0.859)
ROC AUC
P
% sensitivity
% specificity
Cut-off (ng/ml)
0.785 0.765 0.688 0.611 0.598
!0.0001 !0.0001 0.0011 0.0385 0.0453
69.4 83.3 45.2 46.9 62.9
81.7 61.5 88.2 73.0 59.7
215 232.8 66 20.8 998
PH as well as the curve (based on logistic regression scores) for the combination of the three assays. 3.4.2. Discriminating early hepatic fibrogenesis in cystic fibrosis Fig. 6(A) shows the ROC curve for serum PH (AUC 0.814, P!0.0001), which was useful in distinguishing CFLD patients with early fibrogenesis (stages 0 and 1) from those with extensive fibrosis (stages 2–4), with a cut-off level of 59 ng/ml (60% sensitive and 91.7% specific). Serum TIMP-1 showed a trend towards statistical significance in differentiating CFLD patients with no fibrosis (stage 0) from those with fibrosis (AUC 0.725, PZ0.06), with a cut-off at 300 ng/ml (64% sensitive and 83.3% specific) (results not shown). The fit of the model and thus the diagnostic accuracy of assessing CFLD patients with ‘no fibrosis’ vs. ‘fibrosis’ was improved (AUC 0.852, P!0.02) by constructing the ROC curve combining PH and TIMP-1 with logistic regression (Fig. 6B), based on the following equation: PZ
expf8:917 K 0:021XTIMP1 K 0:005XPH g 1 C expf8:917 K 0:021XTIMP1 K 0:005XPH g
P is the probability of inclusion in the ‘fibrosis’ group. A score greater the 0.5 denoting fibrosis in a patient with CFLD (stages 1–4) and a score less than 0.5, indicating no fibrosis (stage 0). Table 4 Diagnostic value of combined serum markers for differentiating patients with CFLD from CFnoLD patients and controls Serum Assay
CLIV and PH CLIV and TIMP-1 TIMP-1 and PH CLIV, TIMP-1 and PH CLIV, TIMP-1 and HA CLIV, TIMP-1, HA and PH all five assays a
ROC AUCa
Logistic regression P
% Sensitivity
% Specificity
% total
0.829 0.807 0.771 0.831
!0.0001 !0.0001 !0.0001 !0.0001
89.8 89.83 86.27 87.76
67.74 66.67 51.61 70.97
81.25 81.05 73.17 81.25
0.805
!0.0001 92.86
65.62
82.95
0.838
!0.0001 89.9
66.67
81.01
0.847
!0.0001 85.71
66.67
78.48
ROC AUC based on logistic regression scores.
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
581
Fig. 5. ROC curves for serum concentrations of CL-IV, TIMP-1 and PH, individually and in combination, for discriminating CFLD patients from those CF patients without liver disease and Controls. The AUC are 0.785 (P!0.0001), 0.765 (P!0.0001), 0.688 (PZ0.0011) and 0.831 (P!0.0001) for CL-IV, TIMP-1, PH and all three combined, respectively.
The logistic model fitted to differentiate ‘early fibrogenesis’ (stages 0 and 1) vs. ‘extensive fibrosis’ (stages 2–4), that included TIMP-1 with PH yielded a ROC AUC of 0.833 (P!0.01) (results not shown). However, this modelling did not significantly improve the diagnostic accuracy when compared to serum PH results alone.
4. Discussion This study evaluated the utility of a number of serum markers of hepatic fibrosis, individually and in combination, in a well characterised cohort of patients with CFLD, where liver biopsies were available for scoring of fibrosis, versus age- and sex-matched patients with CFnoLD and non-CF controls. We demonstrated that serum TIMP-1, PH and CL-IV levels were significantly higher in patients with CFLD compared to CFnoLD and controls, suggesting relative specificity for liver injury in CF. Serum HA was significantly increased in CFLD compared to controls, but not when compared to CFnoLD, indicating the extra-hepatic confounding influences in patients with CF. In addition, serum TIMP-1 and PH were significantly higher in CFLD subjects with minimal or no histological evidence of fibrosis suggesting a potential role for these markers in the early detection of liver injury. When used in combination, serum levels of CL-IV, TIMP-1 and PH demonstrated improved diagnostic accuracy in differentiating CFLD from both CFnoLD and controls, compared to using individual assays. A combination of serum TIMP-1 and PH showed greater diagnostic
Fig. 6. (A) ROC curve for the serum concentration of PH in discriminating patients with early fibrosis (stages 0 and 1) from those with extensive fibrosis (stages 2–4). The area under the curve is 0.814 (P!0.0001). (B). ROC curve combining serum PH and TIMP-1 with logistic regression, for identifying CFLD patients with no fibrosis (stage 0) vs. those with any fibrosis (stages 1–4). The ROC AUC is 0.852 (P!0.02).
accuracy in identifying CFLD patients with no fibrosis (stage 0) from those with fibrosis (stages 1–4). While the pathogenesis of liver disease in CF remains to be determined, we have demonstrated that activated HSC are the cellular source of increased procollagen a1(I) mRNA expression in CFLD and thus are responsible for the hepatic fibrosis and focal biliary cirrhosis seen in CF [8]. Measuring the products of HSC activity less invasively in serum is an attractive proposition for monitoring fibrosis provided the confounding influences of growth and non-hepatic inflammation are minimal. Several studies have attempted to address the utility of serum markers of hepatic fibrosis in CF. Serum collagen VI was studied in 30 children with liver biopsy-verified CFLD,
582
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
133 children with CF and no detectable liver disease (by clinical and conventional blood tests only) and controls [20]. Levels were independent of age, all patients with fibrosis had elevated levels and there was no overlap with controls. However, no stratification was undertaken with severity of fibrosis. Two studies have suggested that human glutathione S transferase A1 (GSTA) was superior to conventional transaminases and comparable to ultrasonography in detecting liver disease, but its utility for detecting hepatic fibrosis was not determined as no biopsies were undertaken [24,25]. Serum PIIIP and PH were studied in 44 children with CF, 26 with suggestion of liver disease by clinical and ultrasound abnormalities [21]. Only four underwent liver biopsy and no comparison of serum markers was made between CFLD and CFnoLD. As a group, children with CF had higher serum PH but not PIIIP verses controls [21]. We related serum markers to histological severity of fibrosis, rather than liver dysfunction, in keeping with other publications. Several patients with only moderate to advanced histological fibrosis had evidence of portal hypertension, a clinical indicator of more advanced liver dysfunction. While our dual-pass biopsy sampling may underestimate the severity of fibrosis in some children, portal hypertension is not synonymous with cirrhosis. Moreover, it seemingly develops earlier in the natural history of CFLD than in other fibrosing liver diseases. The major clinical importance of serum markers remains in the detection and monitoring of hepatic fibrosis before advanced dysfunction becomes evident. TIMP-1 inhibits the activity of MMPs, which degrade extracellular matrix during tissue remodelling associated with active fibrogenesis [26]. TIMP-1 is produced by activated HSC and elevated serum TIMP-I reflects active fibrogenesis in HCV [13], haemochromatosis [14] and alcoholic liver disease [27]. TIMP-1 is active in various organs and disease states, including the failing heart, colitis and asthma [19]. We found serum TIMP-1 significantly elevated in children with CFLD compared to those without liver disease. Using ROC analysis we also showed that serum TIMP-1 might be useful as a diagnostic marker to distinguish between these groups. Early CFLD is associated with marked elevations of TIMP-1, which decreases with progression of fibrosis. This result is similar to previous studies in HCV [13] and haemochromatosis [14] and points to a role for TIMP-1 in remodelling extracellular matrix in early hepatic fibrogenesis in CF. Combining serum TIMP-1 with PH markedly improved the diagnostic accuracy in identifying CFLD patients with fibrosis vs. no fibrosis. PH is an enzyme required for collagen biosynthesis and is produced in the liver by hepatocytes, HSC and endothelial cells. Leonardi and colleagues showed that serum PH was elevated in children with CF vs. non-CF controls, with a non-significant increase in PH levels in four children suspected of having liver disease [21]. Our results show serum PH is elevated in CFLD vs. both CFnoLD
and controls and negatively correlated with hepatic fibrosis scores. This was verified with ROC analysis, demonstrating that serum PH showed high predictive value in distinguishing between patients with early, active hepatic fibrogenesis versus late stage fibrosis, as was previously reported in early alcoholic cirrhosis in adults [9]. This study is the first to show that serum CL-IV is elevated in children with CFLD vs. CFnoLD and controls and thus may identify CF patients with liver disease. Furthermore, when serum levels of CL-IV were used in combination with TIMP-1 and PH, the diagnostic accuracy in identifying patients with CFLD vs. CFnoLD and controls was improved. CL-IV is a major component of basement membrane. Unlike TIMP-1 and PH, serum CL-IV levels were elevated even in more advanced fibrosis. This is not surprising as with fibrosis progression, more CL-IV is degraded by MMPs and the EIA used in this study measures degradation products of CL-IV. Others have also shown sustained elevation in serum CL-IV in chronic liver disease [9,11]. However, contrary to reports in other chronic adult liver diseases [15], CL-IV is not a quantitative marker for progression of fibrosis in CFLD. While we demonstrated that HA was elevated in CFLD vs. controls, there was no significant difference between CFLD and CFnoLD, suggesting HA is less specific for CF liver injury. Elevated serum HA was previously seen in CFLD vs. both CFnoLD and controls [22]. However, as liver biopsy was not performed and no stratification with fibrosis undertaken, CFLD patients may have had more advanced liver disease, which could explain why this study showed a significant difference between CFLD vs. CFnoLD. Others have shown that serum HA is elevated in adult alcoholic liver disease, chronic hepatitis C viral infection and primary biliary cirrhosis [12,28,29]. In these diseases elevated HA levels correlated with the degree of fibrosis. In our study, serum HA did not correlate with histological fibrosis and thus, it is not a useful marker in CFLD. Serum MMP-2 was uninformative in distinguishing CF patients with or without liver disease, from controls. Studies in adults have provided conflicting results on the utility of MMP-2 as a marker of hepatic fibrosis. We previously demonstrated a correlation between serum MMP-2 and fibrosis in haemochromatosis [14]. However, Lichtinghagen and colleagues showed that serum MMP-2 did not correlate with fibrosis in patients with chronic active hepatitis and hepatitis C-induced cirrhosis [30]. These varying data clearly demonstrate the disease-specific utility of individual serum markers. In summary, we have shown that serum TIMP-1, PH and CL-IV are increased in patients with CFLD compared to CFnoLD and control subjects. Serum TIMP-1 and PH levels are elevated in early hepatic fibrogenesis and return to control levels with progression to advanced fibrosis. Serum TIMP-1 and PH may be markers of early events associated with CFLD, prior to significant scar tissue deposition, and thus may be useful in the early detection of liver disease in
T.N. Pereira et al. / Journal of Hepatology 41 (2004) 576–583
CF, particularly when used in combination. We suggest these serum markers require assessment in longitudinal studies of CFLD to determine their utility for predicting significant liver disease and as markers of disease progression.
Acknowledgements This project was funded by a Major Research Project Grant (#913-005 to PJL, JLS, RWS, GAR) and a Seeding Grant (#730 to PJL, JLS, RWS, GAR) from the Royal Children’s Hospital Foundation, Brisbane and by a grant from the National Health and Medical Research Council of Australia (Grant No. 290220 to GAR and PJL).
References [1] Vawter GF, Shwachman H. Cystic fibrosis in adults: an autopsy study. Pathol Annu 1979;14:357–382. [2] Sokol RJ, Durie PR. Recommendations for management of liver and biliary tract disease in cystic fibrosis. Cystic Fibrosis Foundation Hepatobiliary Disease Consensus Group. J Pediatr Gastroenterol Nutr 1999;28:S1–S13. [3] Tanner MS. Liver and biliary problems in cystic fibrosis. J R Soc Med 1992;85:20–24. [4] Williams SG, Westaby D, Tanner MS, Mowat AP. Liver and biliary problems in cystic fibrosis. Br Med Bull 1992;48:877–892. [5] Weber AM, Lenaerts C, Smith L, Roy CC. Screening and diagnosis of cystic fibrosis-related liver disease. Pediatr Pulmonol 1989;Suppl 4: 40–41. [6] Hultcrantz R, Mengarelli S, Strandvik B. Morphological findings in the liver of children with cystic fibrosis: a light and electron microscopical study. Hepatology 1986;6:881–889. [7] Lowe ME, Ameen N, Freedman S, Mullberg A, Werlin SL. Research agenda for pediatric gastroenterology, hepatology and nutrition: cystic fibrosis and pancreatic diseases. Report of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition for the Children’s Digestive Health and Nutrition Foundation. J Pediatr Gastroenterol Nutr 2002;35:S258–S262. [8] Lewindon PJ, Pereira TN, Hoskins AC, Bridle KR, Williamson RM, Shepherd RW, et al. The role of hepatic stellate cells and transforming growth factor-beta(1) in cystic fibrosis liver disease. Am J Pathol 2002;160:1705–1715. [9] Fabris C, Falleti E, Federico E, Toniutto P, Pirisi M. A comparison of four serum markers of fibrosis in the diagnosis of cirrhosis. Ann Clin Biochem 1997;34:151–155. [10] Ueno T, Tamaki S, Sugawara H, Inuzuka S, Torimura T, Sata M, et al. Significance of serum tissue inhibitor of metalloproteinases-1 in various liver diseases. J Hepatol 1996;24:177–184. [11] Castera L, Hartmann DJ, Chapel F, Guettier C, Mall F, Lons T, et al. Serum laminin and type IV collagen are accurate markers of histologically severe alcoholic hepatitis in patients with cirrhosis. J Hepatol 2000;32:412–418. [12] Guechot J, Laudat A, Loria A, Serfaty L, Poupon R, Giboudeau J. Diagnostic accuracy of hyaluronan and type III procollagen amino-terminal peptide serum assays as markers of liver fibrosis in chronic viral hepatitis C evaluated by ROC curve analysis. Clin Chem 1996;42:558–563.
583
[13] Walsh KM, Timms P, Campbell S, MacSween RN, Morris AJ. Plasma levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2) as noninvasive markers of liver disease in chronic hepatitis C: comparison using ROC analysis. Dig Dis Sci 1999;44:624–630. [14] George DK, Ramm GA, Powell LW, Fletcher LM, Walker NI, Cowley LL, et al. Evidence for altered hepatic matrix degradation in genetic haemochromatosis. Gut 1998;42:715–720. [15] George DK, Ramm GA, Walker NI, Powell LW, Crawford DH. Elevated serum type IV collagen: a sensitive indicator of the presence of cirrhosis in haemochromatosis. J Hepatol 1999;31:47–52. [16] Thrailkill KM, Kumar S, Rosenberg CK, Auten KJ, Fowlkes JL. Characterization of matrix metalloproteinases in human urine: alterations during adolescence. Pediatr Nephrol 1999;13:223–229. [17] Bord S, Horner A, Beeton CA, Hembry RM, Compston JE. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) distribution in normal and pathological human bone. Bone 1999;24:229–235. [18] Trivedi P, Risteli J, Risteli L, Tanner MS, Bhave S, Pandit AN, et al. Serum type III procollagen and basement membrane proteins as noninvasive markers of hepatic pathology in Indian childhood cirrhosis. Hepatology 1987;7:1249–1253. [19] Hoshino M, Takahashi M, Takai Y, Sim J. Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma. J Allergy Clin Immunol 1999;104:356–363. [20] Gerling B, Becker M, Staab D, Schuppan D. Prediction of liver fibrosis according to serum collagen VI level in children with cystic fibrosis. N Engl J Med 1997;336:1611–1612. [21] Leonardi S, Giambusso F, Sciuto C, Castigliones S, Castiglione N, La Rosa M. Are serum type III procollagen and prolyl hydroxylase useful as noninvasive markers of liver disease in patients with cystic fibrosis? J Pediatr Gastroenterol Nutr 1998;27:603–605. [22] Wyatt HA, Dhawan A, Cheeseman P, Mieli-Vergani G, Price JF. Serum hyaluronic acid concentrations are increased in cystic fibrosis patients with liver disease. Arch Dis Child 2002;86:190–193. [23] Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991;13:372–374. [24] Hung JC, Howie AF, Beckett GJ, Sood M, Hambleton G, Super M. The use of human glutathione S-transferase A1 in the detection of cystic fibrosis liver disease. J Paediatr Child Health 1998;34:335–338. [25] Sidlova K, Skalicka V, Kotaska K, Pechova M, Chada M, Barlosova J, et al. Serum alpha-glutathione S-transferase as a sensitive marker of hepatocellular damage in patients with cystic fibrosis. Physiol Res 2003;52:361–365. [26] Iredale JP. Tissue inhibitors of metalloproteinases in liver fibrosis. Int J Biochem Cell Biol 1997;29:43–54. [27] Li J, Rosman AS, Leo MA, Nagsˇi Y, Lieber CS. Tissue inhibitor of metalloproteinase is increased in the serum of precirrhotic and cirrhotic alcoholic patients and can serve as a marker of fibrosis. Hepatology 1994;19:1418–1423. [28] Pares A, Deulofeu R, Gimenez A, Caballeria L, Bruguera M, Caballeria J, et al. Serum hyaluronate reflects hepatic fibrogenesis in alcoholic liver disease and is useful as a marker of fibrosis. Hepatology 1996;24:1399–1403. [29] Nyberg A, Engstrom-Laurent A, Loof L. Serum hyaluronate in primary biliary cirrhosis—a biochemical marker for progressive liver damage. Hepatology 1988;8:142–146. [30] Lichtinghagen R, Huegel O, Seifert T, Haberkom CI, Michels D, Flemming P, et al. Expression of matrix metalloproteinase-2 and -9 and their inhibitors in peripheral blood cells of patients with chronic hepatitis C. Clin Chem 2000;46:183–192.