|Year : 2019 | Volume
| Issue : 3 | Page : 972-977
Human Chitinase-3-like Protein amarker of hepatic fibrosis in chronic hepatitis C virus infection
Gehan K El-Saeed1, Rasha I Noreldin1, Ayman A Alghoraieb1, Hanan A Ahmed2
1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Etay El-Baroud Central Hospital, Behira, Egypt
|Date of Submission||02-Jan-2018|
|Date of Acceptance||09-Feb-2018|
|Date of Web Publication||17-Oct-2019|
Hanan A Ahmed
Source of Support: None, Conflict of Interest: None
The aim was to evaluate the level of human chitinase-3-like protein (YKL-40) in patients with hepatitis C virus infection and observe its level in hepatic fibrosis.
Hepatic fibrosis results from long-standing liver damage, and it represents a major health care burden worldwide. In recent years, there has been increasing interest in identifying and describing hepatic fibrosis through the use of noninvasive markers. The serum YKL-40 level has been evaluated as a noninvasive marker of various chronic inflammatory and fibrotic liver diseases, including chronic hepatitis C, chronic hepatitis B, and alcoholic liver disease.
Participants and methods
This study case–control study was carried out in Clinical Pathology Department, Faculty of Medicine, Menoufia University, in the period from September 2015 to November 2016. It was conducted on 77 patients with chronic hepatitis C infection and 10 age-matched and sex-matched healthy individuals as a control group. Serum level of YKL-40 was measured for all the participants of the study by enzyme-linked immune sorbent assay technique.
Mean YKL-40 was lower among control than patient groups (28.0 ± 6.7 and 106.6 ± 38.8, respectively; P < 0.001). Moreover, YKL-40 was lower among mild fibrosis cases than marked fibrosis cases (98.1 ± 41.5 and 111.5 ± 36.6, respectively; P < 0.001).
There is increased serum level of YKL-40 in patients having liver fibrosis than in control group, and its level increases with the degree of fibrosis.
Keywords: hepatitis C infection, liver fibrosis, noninvasive marker
|How to cite this article:|
El-Saeed GK, Noreldin RI, Alghoraieb AA, Ahmed HA. Human Chitinase-3-like Protein amarker of hepatic fibrosis in chronic hepatitis C virus infection. Menoufia Med J 2019;32:972-7
|How to cite this URL:|
El-Saeed GK, Noreldin RI, Alghoraieb AA, Ahmed HA. Human Chitinase-3-like Protein amarker of hepatic fibrosis in chronic hepatitis C virus infection. Menoufia Med J [serial online] 2019 [cited 2020 Apr 4];32:972-7. Available from: http://www.mmj.eg.net/text.asp?2019/32/3/972/268866
| Introduction|| |
Liver fibrosis is caused by an imbalanced remodeling process owing to chronic inflammation associated with excessive scar tissue formation. Hepatic stellate cells, key players in this process, transform from quiescent cells into proliferative, fibrogenic, and contractile myofibroblast-like cells in response to growth factors such as transforming growth factor and platelet-derived growth factor-BB. The differentiation and activation of hepatic stellate cells is tightly controlled by the activity of cAMP. Increased intracellular cAMP levels inhibit fibroblast migration and proliferation and block the phenotype switch into myofibroblasts, leading to less scar tissue formation . Hepatitis C virus (HCV) infection is one of the main causes of chronic liver disease, with estimated 80 million individuals living with chronic hepatitis C worldwide . Human chitinase-3-like protein (YKL-40) (or chitinase-3-like-1, breast regression protein-39, or human cartilage glycoprotein-39) is a chitinase-like protein that is found in humans as a secretion product of particular chondrocytes and synovial cells. The serum YKL-40 level has been evaluated as a noninvasive marker of various chronic inflammatory and fibrotic liver diseases, including chronic hepatitis C, chronic hepatitis B, and alcoholic liver disease. It is secreted by activated macrophages. YKL-40 is believed to act as a chemoattractant for the endothelial cells that can modulate angiogenesis during tissue repair, and it is expressed in multiple tissues including human liver tissue . The aim of this study was to evaluate the level of YKL-40 in patients with chronic HCV infection and observe its level in hepatic fibrosis and its relation with the degree of fibrosis.
| Participants and Methods|| |
This study case–control study was carried in Clinical Pathology Department, Faculty of Medicine, Menoufia University, in the period from September 2015 to November 2016. This study was approved by the Ethical Committee of Faculty of Medicine, Menoufia University. Informed consent was obtained from every patient and control. The study involved 77 patients with HCV infection and 10 age-matched and sex-matched healthy individuals, who served as a control group. All participants underwent the following: thorough history taking; complete clinical examination; abdominal ultrasonography; fibroscan [transient elastography (TE)]; complete blood count performed using XT-1800i hematology analyzer (Sysmex, Kobe, Japan), which is based on fluorescent flow technology and hydrodynamic focusing; liver function tests such as serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), total bilirubin, and direct bilirubin by Cobas Integra 400 autoanalyzer (Roche Diagnostics, Mannheim, Germany); prothrombin time (PT), prothrombin concentration, and international normalized ratio (INR), which were estimated by BFT II Analyzer with the coagulation method (Dade Behring Marburg GmbH, Marburg, Germany); hepatitis viral markers (hepatitis B surface antigen and hepatitis C antibody), which were estimated by the electrochemiluminescence immunoassay 'ECLIA' using Cobas e 411 analyzer (made in Japan; Roche Diagnostics Gmbtt); and serum YKL-40 assessment using enzyme-linked immune sorbent assay (WKEA Med Supplies, New York, New York, USA). We collected 10 ml of blood, which was withdrawn from the patients and the controls under complete aseptic conditions. Each blood sample was divided as follow: 2 ml was collected in vacutainer tube containing EDTA for complete blood count, 1.8 ml was collected in vacutainer tube containing 3.8% citrate solution for PT, and 6 ml was collected in plain vacutainer tube and left to clot for 10–20 min at room temperature. Samples were separated by centrifugation at 3000 rpm for 20 min Clear sera were separated into two aliquots: the first aliquot was separated carefully and kept frozen at −8°C until the time of the assay, and the second aliquot was used for liver functions and viral markers assay.
Enzyme-linked immunosorbent assay for YKL-40 measurement
The assay of YKL-40 level in the samples was done by using purified human YKL-40 antibody coated to microtiter plate wells, which make solid-phase antibody, and then YKL-40 was added to the wells. Thereafter, combined YKL-40 antibody labeled with horseradish peroxidase (HRP) (HRP enzyme-catalyzed) was added, which became an antibody–antigen–enzyme–antibody complex. Washing was done, and then we added 3,3', 5, 5'-Tetramethylbenzidine (TMB) substrate solution. TMB substrate became blue at HRP enzyme-catalyzed. Their action was terminated by the addition of the sulfuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of the YKL-40 in the samples was then determined by comparing the optical density (OD) of the samples to the standard curve.
Data collected were tabulated and analyzed by statistical package for the social sciences (SPSS, version 22; SPSS Inc., Chicago, Illinois, USA) on IBM personal computer. The following statistics were applied: descriptive statistics, for example, percentage, mean, and SD, and analytic statistics, for example, Fischer's exact test, Student's t-test, Mann–Whitney test, analysis of variance (f) test, and Kruskal–Wallis test. P value of less than or equal to 0.05 was considered to be significant.
| Results|| |
In this study, the results of laboratory tests showed that the serum levels of AST, ALT, bilirubin, PT, and INR were higher in the patient group than the control group, with highly significant difference (P < 0.001) [Table 1]. AST level, direct bilirubin level, PT, and INR are significantly increased in patient subgroups based on advancement of liver fibrosis [Table 2]. The mean level for platelet count is lower in the patient subgroups than the control group, with highly statistically significant difference (P < 0.001), but there is no significant difference in the platelet count between the two fibrosis subgroups (P = 0.77). YKL-40 was lower in the control group than the patient subgroups (mean ± SD: 28.0 ± 6.7 and 106.6 ± 38.8, respectively; P < 0.001) [Table 2]. Moreover YKL-40 level was statistically lower among mild fibrosis cases than marked fibrosis cases (mean ± SD: 98.1 ± 41.5 and 111.5 ± 36.6, respectively; P < 0.001) [Table 2] and [Figure 1]. It was found that YKL-40 has the highest diagnostic accuracy with area under the curve (AUC) of 1.00 followed by FIB4 with AUC of 0.992, and finally comes Fibro-Q with the lowest accuracy (AUC: 0.987) [Figure 2], [Figure 3], [Figure 4].
|Table 1: Statistical comparison between the studied groups regarding laboratory investigations and fibrosis markers|
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|Table 2: Statistical comparison between the studied subgroups regarding laboratory investigations and fibrosis markers|
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|Figure 2: Validity of FIB4 for prediction of fibrosis in patient group versus controls.|
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|Figure 3: Validity of Fibro-Q for prediction of fibrosis in patient group versus controls.|
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|Figure 4: Validity of YKL-40 for prediction of fibrosis in patient group versus controls.|
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| Discussion|| |
Fibrosis and cirrhosis of the liver are major causes of morbidity and mortality worldwide and are associated with increasing economic and social effects . Liver fibrosis is a consequence of almost all chronic liver diseases, which is either owing to chronic viral infection by hepatotropic viruses (mainly hepatitis B and C viruses) or autoimmune injury, as well as to metabolic-induced and toxic-induced injury . Chronic infection with HCV is a leading cause of liver-related morbidity and mortality worldwide and predisposes to liver fibrosis and end-stage liver complications . The assessment of liver fibrosis is not only important to determine the prognosis of disease but also to identify patients who require antiviral therapy. For decades, liver biopsy was considered to be the gold standard for staging liver fibrosis. There were two clinically relevant end points: (a) the detection of significant fibrosis (METAVIR F≥2), which indicates that patients should receive antiviral treatment, and (b) the detection of cirrhosis (METAVIR F4), which indicates that patients should be monitored for complications related to portal hypertension and hepatocellular carcinoma . Clinical studies on patients with chronic hepatitis C infection demonstrated that noninvasive methods are accurate for diagnosis and for monitoring liver disease complications in most cases. Moreover, they have a high prognostic value and are cost-effective. Noninvasive methods for assessment of liver fibrosis are gradually being incorporated into new guidelines and are becoming standard of care, which significantly reduces the need for liver biopsy . YKL-40 (also known as human cartilage glycoprotein-39 and chitinase-like protein 1) is a 40 kDa, heparin-binding and chitin-binding glycoprotein and a member of the mammalian chitinase-like proteins. It is secreted from macrophages, neutrophils, and vascular muscle cells. At present, YKL-40 has been proposed to be a new marker of inflammation . YKL-40 is a powerful fibrosis marker with high diagnostic accuracy, in particular in HCV-associated liver disease. Its determination may confirm and improve the diagnostic accuracy of TE especially in early stages of liver fibrosis . In this work, we aimed at the evaluation of the levels of YKL-40 in the serum of chronic HCV-infected patients with different stages of liver fibrosis.
The study included 77 patients distributed as 49 patients with marked degree of fibrosis (F>2) and 28 patients with mild degree of fibrosis (F≤2), and 10 age-matched and sex-matched participants as a control group. The patients were selected from the outpatient clinic of the National Liver Institute, Menoufia University.
In this study the results of laboratory tests showed that the serum levels of AST, ALT, bilirubin, PT, and INR in patients groups were highly significantly higher than the control group. AST level, direct bilirubin level, PT, and INR are significantly increased with the advancement of liver fibrosis. The mean level for platelet count is highly significant lower in patients group than control group, but there is no significant difference in the platelet count between the two fibrosis subgroups. The study aimed at evaluation of noninvasive markers for assessment of liver fibrosis. Among the noninvasive approaches evaluated in this study for the diagnosis of presence or absence of liver fibrosis, it was found that YKL-40 has the highest diagnostic accuracy with AUC of 1.00, followed by FIB4 with AUC of 0.992, and finally came the Fibro-Q with the lowest accuracy and AUC of 0.987. Pichard et al.  showed that mean values of FIB4 increase with advancement of the fibrosis score, from 0.99 ± 0.66 in METAVIR F0 cases to 4.5 ± 2.8 in F4. However, the differences were only statistically significant when groups with severe fibrosis (F3 or F4) were compared with groups with no fibrosis (F0), moderate fibrosis (F1), or intermediate fibrosis (F2). Indeed owing to the rather large overlapping of the results, the FIB4 index could not significantly discriminate F0 from F1 or F2 groups, nor F1 from F2. These were in contrast to our finding, which showed highly significant difference in the median levels of FIB4 between mild and moderate or moderate and severe liver fibrosis. The study by Rath et al. , which included 55 patients with chronic liver disease with 45% of them having chronic HCV viral infection, showed that YKL-40 revealed a clinically relevant association with histologic liver staging and liver stiffness. Patients with significant fibrosis (F2) were compared with those with severe fibrosis or cirrhosis (F3/F4) regarding serum levels of YKL-40. Receiver operating characteristic (ROC) curve analysis for the diagnostic accuracy of a test calculated the highest performance rate for YKL-40 in every fibrosis stage with AUROC values of 0.880 (0.00–1.00) for F >2, 0.854 (0.00–1.00) for F>3, and 0.986 (0.00–1.00) for F = 4, which agreed with our study. Cequera et al.  showed that the line of reference of the hyalouronic acid, YKL-40, and TIMP-1 in the levels combined with other laboratory parameters was significantly associated with the clinical results in 69 patients, with disease progression of 15%. All the baseline levels of the fibrosis markers in serum were significantly associated with histologic fibrosis progression that developed in 70 (33%) of the 209 patients with cirrhosis. However, the hyalouronic acid and platelet counts were better predictors of the histologic progression of fibrosis (AUROC = 0.663). This agreed with levels for hyalouronic acid, TIMP-1, and YKL-40, correspondingly, and this agreed with our study. Younis et al.  showed that serum fibrosis markers were significantly decreased in sustained viral response group as compared with its pretreatment levels for hyalouronic acid, TIMP-1, and YKL-40, correspondingly. This agreed with our study. In conclusion, the present study identified YKL-40 as an inflammatory glycoprotein involved in endothelial dysfunction. It is a noninvasive marker of liver fibrosis. The findings are consistent with many of previously reported studies where its level increased in the serum of HCV-infected patient with the degree of liver fibrosis.
| Conclusion|| |
From this study, we can conclude that YKL-40 is a powerful fibrosis marker with high diagnostic accuracy, particularly in HCV-associated liver disease. Its determination may confirm and improve the diagnostic accuracy of TE, especially in early stages of liver fibrosis, and its level in the serum of chronic HCV-infected patients increases with increase in degree of fibrosis.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Schippers M, Beljaars L, Post E, Lotersztajn S, Reker-Smit C, Han B, et al
. Up regulation of Epac-1 in hepatic stellate cells by prostaglandin E2 in liver fibrosis is associated with reduced fibrogenesis. J Pharmacol Exp Ther 2017; 363
Dolmazashvili E, Abutidze A, Chkhartishvili N, Karchava M, Sharvadze L, Tsertsvadze T. Regression of liver fibrosis over a 24-week period after completing direct-acting antiviral therapy in patients with chronic hepatitis C receiving care within the national hepatitis C elimination program in Georgia: results of hepatology clinic HEPA experience. Eur J Gastroenterol Hepatol 2017; 29
Kumagai E, Mano Y, Yoshio S, Shoji H, Sugiyama M, Korenaga M, et al
. Serum YKL-40 as a marker of liver fibrosis in patients with non-alcoholic fatty liver disease. Sci Rep 2016; 6
Xu R, Zhang Z, Wan F. Liver fibrosis: mechanisms of immune-mediated liver injury. Cell Mol Immunol 2012; 9
Ellis E, Mann D. Clinical evidence for the regression of liver fibrosis. J Hepatol 2012; 56
Sebastiani G, Gkouvatsos K, Pantopoulos K. Chronic hepatitis C and liver fibrosis. World J Gastroenterol 2014; 20:11033–11053.
Castera L. Hepatitis B: are non-invasive markers of liver fibrosis reliable? Liver Int 2014; 34
Xu T, Zhong C, Wang A, Guo Z, Bu X, Zhou Y, et al
. YKL-40 is a novel biomarker for predicting hypertension incidence among pre-hypertensive subjects: a population-based nested case-control study in China. Clin Chim Acta 2017; 472
Rath T, Roderfeld M, Güler C, Wenzel C, Graf J, Beitinger F, et al
. YKL-40 and transient elastography, a powerful team to assess hepatic fibrosis. Scand J Gastroenterol 2011; 46
Pichard A, Mallet V, Nalpas B, Verkarre V, Nalpas A, Dhalluin-Venier V, et al
. FIB-4: an inexpensive and accurate marker of fibrosis in HCV infection. Comparison with liver biopsy and fibro test. J Hepatol 2007; 46
Cequera A, García de León Méndez MC. Biomarkers for liver fibrosis: advances, advantages and disadvantages. Rev Gastroenterol Mé×2014; 79
Younis F, Nafie EA, Abd El Naser G, Samy AKO, Abbass SI, Abo Raia G. YKL-40 as a noninvasive predictor of liver fibrosis after combined interferon/ribavirin therapy in chronic hepatitis C infection. World J Med Sci 2012; 7
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]