Available online at

Transcription

1 Available online at Annals of Clinical & Laboratory Science, vol. 45, no. 6, Galectin-3 Concentration in Liver Diseases Monika Gudowska 1, Ewa Gruszewska 1, Bogdan Cylwik 2, Anatol Panasiuk 3, Magdalena Rogalska 3, Robert Flisiak 3, Maciej Szmitkowski 1, and Lech Chrostek 1 1 Department of Biochemical Diagnostics, Medical University of Bialystok, 2 Department of Pediatric Laboratory Diagnostics, Medical University of Bialystok, and 3 Department of Infectious Diseases and Hepatology, Medical University of Bialystok, Bialystok, Poland Abstract. The aim of this study was to evaluate the changes in serum galectin-3 concentration during liver diseases. The results were compared with hyaluronic acid concentration in combination with prothrombin index as a HAPRI index. Serum samples were obtained from 20 healthy volunteers and 109 patients suffering from alcoholic cirrhosis (AC) - 57, non-alcoholic cirrhosis (NAC) - 30 and toxic hepatitis (HT) - 22 patients. Cirrhotic patients were classified according to Child-Pugh scale. Galectin-3 concentration was measured by the chemiluminescent microparticle immunoassay and HAPRI index was calculated using a formula. There was a significant increase in the serum concentration of galectin-3 in patients with AC, NAC and HT, and significant differences between diseases were observed. The mean galectin-3 concentration in AC was significantly higher than that in HT. The serum galectin-3 level differs significantly according to severity of cirrhosis and was the highest in Child-Pugh class C. The sensitivity, specificity, accuracy, positive and negative predictive values and the area under the ROC curve for galectin-3 and HAPRI were high and similar to each other. In conclusion, galectin-3 is a good marker of fibrosis in cirrhosis and toxic hepatitis, which reflects the stage of liver damage, like the HAPRI index. Introduction In the past decade many clinical and laboratory researches were directed to develop non-invasive methods for diagnosing hepatic fibrosis. Promising biomarkers of fibrosis were evaluated singly and in combination for their diagnostic utility. As noted in current reviews the capacity of serum biomarkers to detect the presence or absence of significant hepatic fibrosis/cirrhosis is well established, but they are inadequate to differentiate between intermediate stages of fibrosis. More specific biomarkers are needed to improve diagnostic accuracy [1]. Galectin-3 is one of the most recently identified markers of liver fibrosis [2]. It has been reported that this β-galactoside-binding lectin is a regulator of the phagocytic process via cross-linking and enhancing the tethering of apoptotic cells. During the process of phagocytosis of apoptotic hepatocytes, hepatic stellate cells (HSC) differentiate into myofibroblasts [3]. Moreover, galectin-3 stimulates the myofibroblasts to produce procollagen I, which Address correspondence to Lech Chrostek, Department of of Biochemical Diagnostics, Medical University of Bialystok, Waszyngtona 15A St., Bialystok, Poland; phone: ; fax: ; e mail: chrostek@umb.edu.pl irreversibly cross-links to form collagen. Galectin-3 stimulated myofibroblasts may also produce transforming growth factor-β1, which regulates fibrogenesis [2,3,4] as HSC from mice lacking galectin-3 gene displayed reduced expression of α-smooth muscle actin and decreased phagocytic activity [3]. A significant increase of galectin-3 concentration in the blood serum is observed in many pathological processes which take place in various tissues [5]. In human and mice livers, the presence of galectin-3 was detected in the bile duct epithelial and Kupffer cells, but the expression was absent in normal hepatocytes [5,6]. An increased expression of galectin-3 was observed in proliferating fibroblasts, mainly in the nucleus [5]. The elevated expression of this lectin was confirmed at mrna level [4]. Genetic studies have shown that disruption of galectin-3 gene blocks myofibroblasts emphatically attenuating the process of liver fibrosis. This data suggests that galectin-3 is required for increased cellular matrix production and for liver damage [2,5]. Because galectin-3 directly affects the process of phagocytosis and fibrosis, levels of this lectin can reflect the progression of liver damage. In this study, galectin-3 concentration was examined in cirrhosis /15/ by the Association of Clinical Scientists, Inc.

2 670 Annals of Clinical & Laboratory Science, vol. 45, no. 6, 2015 Figure 1. Serum levels of galectin-3 and HAPRI index in liver diseases. C, controls; AC, alcoholic cirrhosis; NAC, nonalcoholic cirrhosis; HT, toxic hepatitis. (alcoholic and non-alcoholic origin) and toxic hepatitis, the liver disorders with different stages of fibrosis. The results for galectin-3 were compared with the best non-invasive fibrosis markers hyaluronic acid and prothrombin time, encapsulated in one test named HAPRI. HAPRI is not related to liver inflammation because it is independent in variation of aminotransferases levels [7]. It was proven that concentration of HA increases in liver diseases and directly reflects the stage of fibrosis and correlates with clinical severity of disease [7,8]. Therefore, the aim of this study was to assess the changes in galectin-3 concentration, as a marker of liver fibrosis, during alcoholic and non-alcoholic cirrhosis and toxic hepatitis. The results were compared with the best non-invasive test of liver fibrosis which is HA in combination with INR as a HAPRI index. Materials and Methods The tested group consisted of 109 patients consecutively admitted to the Department of Infectious Diseases and Hepatology (Medical University of Bialystok). The patients tested were males (74) and females (35) aged between years. Patients were divided into 3 subgroups: alcoholic cirrhosis (AC) - 57 patients, non-alcoholic cirrhosis (NAC) - 30 patients and toxic hepatitis (HT) - 22 patients. Non-alcoholic cirrhosis was caused by chronic hepatitis C -13 patients, chronic hepatitis B - 1, autoimmune hepatitis - 1, primary biliary cirrhosis - 4 and by undefined factors The severity of liver cirrhosis was evaluated by Child-Pugh scores (score A - 27, score B - 31 and score C - 25). The diagnosis was based on the signs, symptoms, physical exams, biochemical liver panel and results of ultrasound or computed tomography (CT) scan of the abdomen. To confirm the diagnosis of HCV, anti- HCV test was performed. Also, all patients were interviewed regarding their use of alcohol. The acute alcohol

3 Galectin-3 in liver diseases 671 Table 1. Diagnostic usefulness of galectin-3 and HAPRI in liver diseases. Liver disease Cut-off Sensitivity Specificity ACC PPV NPV (from ROC) [%] [%] [%] [%] [%] Galectin-3 AC ng/ml NAC ng/ml HT ng/ml HAPRI AC NAC HT ACC, diagnostic accuracy; PPV, positive predictive value; NPV, negative predictive value; AC, alcoholic cirrhosis; NAC, non-alcoholic cirrhosis; HT, toxic hepatitis. abuse was the cause of seven cases of toxic hepatitis. All patients underwent heart ultrasonography to exclude cardiomyopathy. The control group consisted of 20 healthy volunteers (11 males and 9 females) recruited from hospital workers. All subjects (healthy and sick) gave their informed consent to participate in the studies. The study was approved by the Bioethical Committee at the Medical University in Bialystok. Blood samples for biochemical analyses were obtained from a peripheral vein after admittance and before treatment. The sera were separated by centrifugation and stored at -86 o C until assayed. Besides serum, a part of each blood sample was collected into tubes containing 3.8% liquid sodium citrate for hemostasis analyses. Galectin-3 concentration was measured according to the chemiluminescent microparticle immunoassay on the Architect ci8200 (ARCHITECT Galectin-3, Abbott, Germany). Hyaluronic acid (HA) was determined on the same analyzer by immunochemical method using WAKO reagents. Prothrombin time (PT) was determined on STA Compact Max analyzer (Stago) by viscometric method. HAPRI index was calculated on the basis of the following formula: (HA [ng/l]/inr)x100. Statistical analysis. The differences between tested and control groups were evaluated by Mann-Whitney U test. To calculate the correlation between variables, Spearman s rank correlation coefficient was used. To test the hypothesis about the differences between liver diseases, ANOVA rank Kruskal-Wallis test was performed. We considered P-values <0.05 as statistically significant. The diagnostic performance of each test was calculated as sensitivity, specificity, positive and negative predictive values (PPV, NPV), and accuracy. The diagnostic values of the tests were compared by the area under the receiver operating characteristic (AUROC) curve. Results The mean values of galectin-3 and HAPRI according to the liver disease and severity of liver cirrhosis are presented in Figure 1. The serum galectin-3 concentration was significantly elevated in alcoholic and non-alcoholic cirrhosis, and in toxic hepatitis group (mean±sd; 23.42±11.78, 18.14±6.99, 16.88±5.46 ng/ml, respectively) in comparison to the control group (9.90±2.21 ng/ml; P<0.001 for all comparisons). There were significant differences in the serum galectin-3 levels between liver diseases (ANOVA: H=8.942, P=0.011). Further analysis showed that the mean galectin-3 concentration in AC was significantly higher than that in HT group (P=0.034). Also, the values of non-invasive fibrosis indicator HAPRI were significantly increased in AC, NAC and HT (71.922±68.232, ±27.452, ±20.834) compared to controls (3.677±1.032; P<0.001 for all comparisons). The analysis of variance revealed that liver diseases affect the value of HAPRI (H=21.490, P<0.001). Post-hoc analysis showed that the values of HAPRI were higher in AC group in comparison to NAC and HT group (P=0.030, P<0.001, respectively). The correlation between galectin-3 concentration and HAPRI was assessed using Spearman s rank correlation test (R=0.555, P=0.000). Galectin-3 concentration correlated with HAPRI in non-alcoholic cirrhosis and toxic hepatitis (R=0.429, P=0.018; R=0.439, P=0.041, respectively), but galectin-3 concentration did not correlate with HAPRI in alcoholic cirrhosis (R=0.102; P=0.450).

4 672 Annals of Clinical & Laboratory Science, vol. 45, no. 6, 2015 Figure 2. The ROC curves of serum galectin-3 and HAPRI index in liver diseases. The Spearman s correlation analysis demonstrated an association between severity of liver cirrhosis (Child-Pugh scale) and galectin-3 concentration (R=0.367, P<0.01) and also with HAPRI values (R=0.591, P<0.01). The analysis also showed that galectin-3 concentration correlated with the severity of liver cirrhosis in NAC patients (R=0.412, P=0.030), but the HAPRI correlated with the severity of liver cirrhosis in AC patients (R=0.493, P<0.01). ANOVA rank Kruskal-Wallis analysis confirmed that the severity of liver cirrhosis affects the galectin-3 and HAPRI level (H=12.816, P=0.002; H=28.284, P<0.001, respectively). Further analysis showed that the values of galectin-3 were higher in Child-Pugh B and C (23.72±11.29, 24.00±11.20 ng/ml, respectively) scores than that in Child-Pugh A score (17.20±6.56 ng/ml) (P<0.05 for both). Additionally, the values of HAPRI were higher in Child-Pugh C score (125.98) than that in Child-Pugh B and A scores (68.943, P=0.014; , P<0.001, respectively). Also, there were significant differences in HAPRI values between Child-Pugh A and B score (P=0.021). The diagnostic value of galecin-3 and HAPRI index are presented in Table 1. Galectin-3 and HAPRI had the highest predictive value for the diagnosis of alcoholic cirrhosis at the cut-off points ng/ ml and 64.53, respectively. Both tests had the sensitivity equal to 96.5%, but HAPRI has a specificity higher than galectin-3 in the case of AC. The diagnostic accuracy (ACC), PPV, NPV values for HAPRI and galectin-3 were similar to each other. The value of AUC ROC of HAPRI in case of alcoholic- and non-alcoholic cirrhosis is higher than galectin-3, but in diagnosis of toxic hepatitis galectin-3 has better diagnostic power than HAPRI (Figure 2). Discussion In this study, we tried to specify the diagnostic value of the new fibrosis indicator, galectin-3, in patients with liver diseases. We have shown that the mean serum level of galectin-3 is elevated in patients with liver injury. The changes in galectin-3 concentration may originate from increased liver fibrogenesis, in which galectin-3 directly participates [2]. Among the studied diseases, the highest galectin-3 concentration was observed in alcoholic cirrhosis (over two times higher than that in the control group), but the lowest in toxic hepatitis. These differences may result from different mechanisms of liver injury. Existing data suggest that the role of galectin-3 is different in inflammation (such a toxic hepatitis) than in the mechanisms involved in cirrhosis [2]. Expression of galectin-3 can also differ depending on the intensity of disease process and its chronicity. Therefore, it is important to take into account the fact, that advanced cirrhosis is a result of prolonged, multiannual process of chronic injury and fibrosis, but toxic hepatitis is an acute and very intense process. Upon tissue injury, galectin-3 which is normally stored in the cytoplasm, is actively secreted to the blood and biological fluids by activated and damaged cells [2,3]. Studies based on the carbon tetrachloride (CCl4) as an inducer of toxic injury demonstrated that apoptosis or/and necrosis are the initiating events of fibrosis, which

5 Analytic Performance of DongJiu 673 may result in elevated expression of galectin-3 [3]. The expression of galectin-3 was increased especially at the periphery of the inflamed damaged areas and in myofibroblasts. It has been documented that galectin-3 concentration is low in normal human liver and dramatically increases in regenerating cirrhotic nodules, especially at the periphery as a probable result of high mitotic rate [5]. It is assumed that in normal liver hepatic stem cells express the galectin-3. In cirrhotic liver these stem cells undergo rapid proliferation and create new hepatocytes. Finally, regenerating nodules in cirrhotic liver express more galectin-3. Hsu et al. proved that liver cirrhosis and hepatocellular carcinoma frequently expressed significant levels of galectin-3 [5]. It means that galectin-3 participates in cell growth and neoplastic transformation. We speculated that very high galectin-3 concentrations in alcoholic cirrhosis in our studies are probably caused by a combination of two different mechanisms of liver injury: the first caused by alcohol leading to inflammation and the second one involved in long-term process of fibrogenesis. The correlation study demonstrated an association between severity of liver cirrhosis (Child-Pugh scale) and galectin-3 concentrations. The results showed that the galectin-3 concentrations were the highest in the most severe stage of liver injury (Child-Pugh class C). This might be associated with the intense hepatic stem cells proliferation and the expression of galectin-3. It is possible that proliferating cells expressing galectin-3 are in a process of being transformed, thus indicating an early tumor development [5]. Our study showed that galectin-3 has the highest diagnostic sensitivity, ACC, PPV and NPV in alcoholic cirrhosis. Diagnostic specificity was the same in all liver diseases 90%. For comparison, HAPRI index has 100% specificity and PPV. It is a consequence of the lack of false positive results. The higher values of HAPRI than that of galectin-3 are associated with the presence of hyaluronic acid encapsulated in HAPRI formula. Hyaluronic acid is the best individual test reflecting the degree of hepatic fibrosis [9]. Previous study has demonstrated that hyaluronic acid concentration included in HAPRI index increases with the development of liver fibrosis and correlates with clinical severity of cirrhosis [7,8]. Our study demonstrated an association between HAPRI value and the stage of liver injury. HAPRI was 4-times increased in the class C in comparison to class A, and multifold increased in comparison to healthy subjects. The lowest values of HAPRI index were observed in HT. This result seems to be associated with the normal values of INR. In summary, galectin-3 had a very high diagnostic power, similar to that of to HAPRI (area under the ROC curve), in the diagnosis of alcoholic and non-alcoholic cirrhosis and toxic hepatitis. A major conclusion is that the mean values of galectin-3 in liver diseases are elevated but different, and related to severity of liver damage. Galectin-3 has a high diagnostic power in diagnosis of alcoholic and non-alcoholic cirrhosis and toxic hepatitis, like the HAPRI index. Measurements of galectin-3 concentrations could be an excellent indicator of liver fibrosis, because it is easy to use, simple, quick and is a non-invasive test. References 1. Fallatah HI. Noninvasive biomarkers of liver fibrosis: An overview. Advances in Hepatology 2014; ID dx.doi. org/ /2014/ Henderson NC, Mackinnon AC, Farnworth SL, Poirier F, Russo FP, Iredale JP, Haslett C, Simpson KJ, Sethi T. Galectin-3 regulates myofibroblast activation and hepatic fibrosis. Proc Natl Acad Sci U S A 2006;103: Jiang JX, Chen X, Hsu DK, Baghy K, Serizawa N, Scott F, Takada Y, Takada Y, Fukada H, Chen J, Devaraj S, Adamson R, Liu FT, Török NJ. Galectin-3 modulates phagocytosis-induced stellate cell activation and liver fibrosis in vivo. Am J Physiol Gastrointest Liver Physiol 2012;302:G Wijesundera KK, Juniantito V, Golbar HM, Fujisawa K, Tanaka M, Ichikawa C, Izawa T, Kuwamura M, Yamate J. Expressions of Iba1 and galectin-3 (Gal-3) in thioacetamide (TAA)-induced acute rat liver lesions. Exp Toxicol Pathol 2013;65: Hsu DK, Dowling CA, Jeng KC, Chen JT, Yang RY, Liu FT. Galectin-3 expression is induced in cirrhotic liver and hepatocellular carcinoma. Int J Cancer 1999;81: Nomoto K, Nishida T, Nakanishi Y, Fujimoto M, Takasaki I, Tabuchi Y, Tsuneyama K. Deficiency in galectin-3 promotes hepatic injury in CDAA diet-induced nonalcoholic fatty liver disease. ScientificWorldJournal 2012;2012: Crisan D, Radu C, Grigorescu MD, Lupsor M, Feier D, Grigorescu M. Prospective non-invasive follow-up of liver fibrosis in patients with chronic hepatitis C. J Gastrointestin Liver Dis 2012;21: Halfon P, Bourlière M, Pénaranda G, Deydier R, Renou C, Botta-Fridlund D, Tran A, Portal I, Allemand I, Rosenthal- Allieri A, Ouzan D. Accuracy of hyaluronic acid level for predicting liver fibrosis stages in patients with hepatitis C virus. Comp Hepatol 2005;4:6. 9. Kelleher TB, Afdhal N. Noninvasive Assessment of Liver Fibrosis. Clin Liver Dis 2005;9: