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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 44  |  Issue : 2  |  Page : 124-127

Study of m65 hepatocyte death marker in multitransfused patients with β-thalassemia major in upper Egypt


1 Department of Clinical Pathology, Faculty of Medicine, Aswan University, Aswan, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt

Date of Submission02-Dec-2018
Date of Acceptance10-Jan-2019
Date of Web Publication15-Nov-2019

Correspondence Address:
HebatAllah Abdellatif
Department of Clinical Pathology, Faculty of Medicine, Aswan University, Aswan 81528
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.271082

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  Abstract 


Introduction Iron overload is a common complication in patients with β-thalassemia major (β-TM) and is defined as serum ferritin level higher than 1000 ng/ml. Deposition of iron in body organs, mainly in the liver, leads to hepatocytes damage. Keratins are the major epithelial-specific subgroup of intermediate filament proteins. Increased apoptosis and/or necrosis play a role in the pathogenesis and determination of disease progression. Keratin 18 is cleaved by caspases during apoptosis and creates M30 fragments. Soluble intact K18 (M65) is also released from cells during cell death.
Patients and methods A prospective case–control study of 70 multitransfused patients with β-TM and 22 controls was conducted in Paediatric Haematology Unit of Assiut University hospital from January 2016 till April 2017. The study participants were screened for liver enzymes, hepatitis markers (hepatitis C virus antibodies and hepatitis B virus antigen), and serum ferritin levels. Circulating levels of human keratin 18-M65 (K18-M65) were measured by enzyme-linked immunosorbent assay.
Results Serum M65 levels were higher in patients’ group compared with healthy controls (P<0.001). M65 serum levels were also positively correlated with the serum levels of liver transaminases including aspartate aminotransferase (AST) (δ-0.470, P<0.001) and alkaline phosphatase (δ=0.180, P=0.043), and serum ferritin (δ=0.450, P=0.043).
Conclusion M65 serum levels increased significantly with increased levels of serum ferritin and liver enzymes (aspartate aminotransferase and alkaline phosphatase) between β-TM patient groups.

Keywords: keratin, M65, β-thalassemia major


How to cite this article:
Abdellatif H, NasrEldin E, Galal SH, Ibrahim MA. Study of m65 hepatocyte death marker in multitransfused patients with β-thalassemia major in upper Egypt. Egypt J Haematol 2019;44:124-7

How to cite this URL:
Abdellatif H, NasrEldin E, Galal SH, Ibrahim MA. Study of m65 hepatocyte death marker in multitransfused patients with β-thalassemia major in upper Egypt. Egypt J Haematol [serial online] 2019 [cited 2019 Dec 5];44:124-7. Available from: http://www.ehj.eg.net/text.asp?2019/44/2/124/271082




  Introduction Top


Iron overload is the commonest complication of patients with β-thalassemia major (β-TM) and defined as serum ferritin level more than 1000 ng/ml. Patients with β-TM receive iron chelation to reduce iron overload; however, iron deposition in the organs is still the cause of tissue damage [1].

Liver biopsy has many complications as it is an invasive technique, and the sample size of liver biopsy specimen is small, as it represents ∼1/50 000 of total liver mass that may lead to technical errors, which limit interpretations. So, attention has focused on the identification of noninvasive biomarkers such as the cytoskeletal components, including keratins [1].

Keratins, formerly known as cytokeratins, are the upmost epithelial-specific subgroup of intermediate filament proteins that are useful for detecting apoptosis and necrosis. Keratin 18 is a type 1 K which is the most keratins in epithelial cells (e.g. hepatocytes and intestinal cells). Soluble intact K18 (M65) is released from dying cells. Apoptosis and/or necrosis have a role in pathogenesis of liver diseases and determination of disease progression. So, markers of hepatocyte apoptosis or necrosis can be used to monitor liver diseases. During liver cell apoptosis, K18 is cleaved by activated caspases, and this can be detected in serum or plasma by the M65 (it detects caspase-cleaved and uncleaved K18) [1].

In this study, we aimed to estimate M65 levels in serum of patients with β-TM and its relation with serum ferritin and liver enzyme levels to study its possible effect on liver injury due to iron deposition.


  Patients and methods Top


Patients

This prospective case–control study was recruited from Paediatric Haematology Unit of Assiut University hospital in the period from January 2016 to April 2017 on 70 blood transfusion-dependent patients with β-TM on regular iron chelation therapy (40 males and 30 female). Their ages ranged from two to 18 years old. Moreover, 22 apparently healthy children were included as controls. Diagnosed as transfusion-dependent β-TM was done according to criteria of Thalassaemia International Federation [2]. The study was approved by the Scientific and Ethical Committees at Faculty of Medicine, Assiut University. Informed consents were obtained from all participants’ guardians according to the Declaration of Helsinki.

Blood was collected from patients aseptically. A volume of 3-ml blood was collected in plain tubes, separated in sterile aliquots, and stored at −20°C until use in estimation of serum ferritin and liver enzymes levels on Modular P auto analyser (Roche Diagnostics, Mannheim, Germany) and hepatitis markers [antihepatitis C virus antibodies (anti-HCVAb) and HBsAg] done on ARCHITECT i1000sr Immunology Analyzer (Abbott, Germany). Human keratin 18-M65 (K18-M65) enzyme-linked immunosorbent assay kit (catalog no. SG-10749; SinoGene Clon Biotech Co. Ltd, China) was used according to the manufacturer’s instructions for quantitative measurement of M65. This was performed in Laboratory of Clinical Pathology Department, Assiut University Hospital, Assiut University, Egypt.

Statistical analysis

The data analysis was made using the statistical package for the social sciences for Windows statistical software (version 16, SPSS: an IBM Company, version 16.0; IBM Corporation, Armonk, New York, USA). Data are presented as means±SD, median, range, or percentage where appropriate. The independent t-test was used to compare mean among quantitative data, whereas Spearman’s correlation coefficient were used to compare qualitative data. Values of P less than 0.05 were regarded as a significant result.


  Results Top


Seventy patients with β-TM and 22 healthy controls were included in our study. Their demographics are illustrated in [Table 1]. Both ALT and aspartate aminotransferase (AST) levels were higher in patient group than controls, with statistically significant differences between groups (P<0.001). There is a statistical significance difference in serum alkaline phosphatase (ALP) levels between the study groups (P=0.027).
Table 1 Demographic data and comparative analysis between patients with β-thalassemia major and controls regarding hematologic data and liver enzymes

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Fifty patients with β-TM had negative HCVAbs, whereas 20 patients had positive HCVAbs. The mean serum M65 levels were higher in patients negative for hepatitis C than patients who were positive, with nonsignificant statistical difference between groups (P=0.0514) ([Table 2]).
Table 2 Comparison of M65 levels between hepatitis C virus antibodies positive and negative β-thalassemia major

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Serum M65 levels were higher in the patient group than controls, with statistically significant difference between both (P<0.001) ([Table 3]).
Table 3 Comparison of M65 levels between cases and controls

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There was moderate significant correlation between M65 and both serum of AST and ferritin levels (δ=0.470 and 0.450, respectively), with significant P value of less than 0.001 and 0.043, respectively. Weak significant correlation (δ=0.180) was found between M65 and ALP (P=0.043), whereas insignificant weak correlation (δ=0.192) was found between M65 and ALT (P=0.056) ([Table 4]).
Table 4 Correlation between circulating levels hepatocyte cell death marker (M65) and serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and ferritin in β-thalassemia major

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  Discussion Top


Iron overload is highly damaging to hepatocytes. As soon as the protective effect of the iron storage protein ferritin is exceeded, hepatocyte damage occurs leading to cellular necrosis (biologically expressed by increase serum transaminase activities: alanine aminotransferase and AST) followed by the progressive development of fibrosis, the ultimate stage of which is cirrhosis [3].

The way iron toxicity develops is largely dependent upon the level of plasma nontransferrin-bound iron. Indeed, part of this iron species is in the form of labile plasma iron which has a high propensity to produce reactive oxygen species (ROS) [4],[5]. These are known to damage membrane lipids, affecting not only hepatocyte plasma membranes but also the membranes of intracellular organelles, including cell nuclei [2].

Cytoskeletal components such as keratins (Ks), are considered of the intermediate filament family [6]. Keratin 18 is a type-I K present in epithelial cells (e.g. hepatocytes and intestinal cells) [7]. Uncleaved K-18 (M65) reflects many forms of liver cell death, including necrosis, apoptosis, necroptosis, autophagy, and so on. K18 is detected by using the M65 antibodies [8].

Serum M65 and M30 are reported as valuable indicators of liver inflammation. M65 is better than M30 in identifying inflammation of liver especially in patients with normal ALT. Moreover, M65 was important in the staging of liver fibrosis and M30 had no role in detection of liver fibrosis progress [8]. We analyzed the M65 antibodies in 70 patients with β-TM and 22 controls by using enzyme-linked immunosorbent assay.

Serum M65 levels were significantly higher in patients with β-TM than controls with P value less than 0.001. There is a possibility that iron overload induces generation of ROS in liver cells, which cause activation of caspases and apoptosis (mitochondrial pathway).

The level of M65 was higher in patients with β-TM than healthy participants in the study done in Iran. This suggested that necrosis is probably more prominent in hepatocyte death mode of patients with β-thalassemia than apoptosis [9]. Mitochondrial-dependent apoptosis may cause necrosis, and when ATPs wane, this might lead to necrosis instead of apoptosis [10].

Accumulation of iron-induced lipid ROS in hepatocytes may be another possibility, which results in occurrence of ferroptosis (regulated cell death depends on iron), which differ from apoptosis and necrosis. Thus, releasing M65 in circulation is possibly related to necroptosis or ferroptosis [11].

The study by Younossi et al. [12] reported that M65 and M30 identify effectively patients with hepatic fibrosis from healthy people and differentiate mild, moderate, as well as sever hepatic fibrosis. Fibrosis in chronic hepatitis C in addition was found to be related to M65 and M30 [13] and primary biliary cirrhosis [14]. Some other studied failed to found a relationship between M65 and fibrosis [15],[16].

Our results found a moderate significant correlation between M65 and serum AST and ferritin levels (δ=0.470 and 0.450, respectively). However, weak insignificant correlation (δ=0.192) was found between M65 and ALT (P=0.056). Moreover, weak significant correlation was found between M65 and ALP (P=0.043).

To the best of our knowledge, a previous study done on patients with thalassemia found that there is no correlation between M65 levels and liver enzymes. Moreover, the authors detected that the serum levels of M65 were not correlated with serum levels of ferritin, transferrin, hepcidin, and ALP in patients with β-thalassemia [9].

HCV is a common transfusion-related complication in patients with β-TM. We found nonsignificant association in mean M65 serum levels between patients having HCV positive and HCV negative results. Data showed that M65, but not M30, increased with increased severity of fibrosis [8]. These results were similar to a previous study on M65 which was better than M30 in diagnosis of fibrosis [12]. It is known that dead hepatic cells provide the key stimuli of fibrosis. In addition, the dying (but viable) hepatocytes also release fibrosis-inducing factors. So, we can understand why M65 was more accurate than M30 in inflammation and fibrosis prediction. They also found that M65 and M30 levels were correlated with inflammation in patients with the same stage of fibrosis, but not correlated with fibrosis in patients with the same grade of inflammation. So, M65 was a direct indicator of inflammation and an indirect indicator of fibrosis [8].In our results, higher M65 levels was detected in patients with β-TM with weak insignificant correlation with ALT suggesting that M65 is a good indicator for prediction of early liver affection in patients on regular blood transfusion even before increase in ALT levels. So, early iron chelation therapy should be recommended in patients with high M65 levels. Hydroxyurea may benefit patients with β-TM with high M65 levels as it stimulates γ-globin gene expression in vivo and therefore reduces α-chain accumulation and subsequently, decreased the severity of clinical symptoms [17].


  Conclusion Top


β-TM showed higher circulating levels of M65 than healthy controls. M65 serum levels are positively correlated to serum ferritin and liver enzymes levels (AST and ALP) between β-TM patient groups. We can assess from the results the possibility that M65 has an effect on liver injury, and it can be used in assessment of liver fibrosis instead of invasive liver biopsy. Future studies should focus on the role of M65 and M30 in assessment of liver fibrosis degree and in follow-up of its progression.

Acknowledgements

H.A., E.N., Sh.H.G., and M.A.I. participated in research design and carried out the research. H.A. and E.N. conducted the data analysis and wrote the paper.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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2.
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Hershko C. Pathogenesis and management of iron toxicity in thalassemia. Ann N Y Acad Sci 2010; 1202:1–9.  Back to cited text no. 5
    
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Yilmaz Y, Dolar E, Ulukaya E, Akgoz S, Keskin M, Kiyici M et al. Soluble forms of extracellular cytokeratin 18 may differentiate simple steatosis from nonalcoholic steatohepatitis. World J Gastroenterol 2007; 13:837–844.  Back to cited text no. 7
    
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Nafiseh E, Behjat M, Marjan G, Reza MM. Elevated serum levels of cell death circulating biomarkers, M30 and M65, in patients with β-thalassaemia major. Haemoglobin 2013; 37:404–410.  Back to cited text no. 9
    
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Jaeschke H, Lemasters JJ. Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury. Gastroenterology 2003; 125:1246–1257.  Back to cited text no. 10
    
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Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012; 149:1060–1072.  Back to cited text no. 11
    
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Younossi ZM, Page S, Rafiq N, Birerdinc A, Stepanova M, Hossain N et al. A biomarker panel for non-alcoholic steatohepatitis (NASH) and NASH-related fibrosis. Obes Surg 2011; 21:431–439.  Back to cited text no. 12
    
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Parfieniuk-Kowerda A, Parfieniuk-Kowerda A, Lapinski TW, Rogalska-Plonska M, Swiderska M, Panasiuk A et al. Serum cytochrome c and m30-neoepitope of cytokeratin-18 in chronic hepatitis C. Liver Int 2014; 34:544–550.  Back to cited text no. 13
    
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Sekiguchi T, Umemura T, Fujimori N, Shibata S, Ichikawa Y, Kimura T et al. Serum cell death biomarkers for prediction of liver fibrosis and poor prognosis in primary biliary cirrhosis. PLoS One 2015; 10:e0131658.  Back to cited text no. 14
    
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Papatheodoridis GV, Hadziyannis E, Tsochatzis E, Chrysanthos N, Georgiou A, Kafiri G et al. Serum apoptotic caspase activity as a marker of severity in HBeAg-negative chronic hepatitis B virus infection. Gut 2008; 57:500–506.  Back to cited text no. 15
    
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Bae CB, Kim SS, Ahn SJ, Cho HJ, Kim SR, Park SY et al. Caspase-cleaved fragments of cytokeratin-18 as a marker of inflammatory activity in chronic hepatitis B virus infection. J Clin Virol 2013; 58:641–646.  Back to cited text no. 16
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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