Spectrum of Cortactin Isoforms in the Urine of Patients with Liver Cirrhosis and Their Diagnostic Value as a Potential Molecular Marker of its Severity

Abstract

Introduction. Non-invasive methods of research of the human organism clinical conditions have been widely used in medicine as a diagnostic and prognostic test. Since the protein content of human urine is closely related to the peculiarity of the functioning of biological systems, its qualitative and quantitative characteristics are promising markers for control of various processes in organism. An important source of protein markers is the results of detailed proteomics (two-dimensional electrophoresis of proteins followed by mass spectrometry) of human urine, depending on the disease. Recently, the protein of SRC8 (Sarc family protein kinase substrate or cortactin) was detected among 12 new proteins in urine protein analyzes in breast cancer patients. The aim of the study. To investigate the spectrum of cortactin isoforms in the urine of patients with liver cirrhosis (LC) and their diagnostic value as a potential molecular marker of its severity. Materials and methods. In a randomized pre-stratified method, 18 patients (women – 41.7 %, males – 58.3 %) of 54.8 ± 2.5 years old who were treated at Lviv Regional Hepatological Center based on the department of Internal Medicine №1 of Danylo Halytsky Lviv National Medical University and Gastroenterological Department of the Communal Nonprofit Enterprise of the Lviv Regional Council ″Lviv Regional Clinical Hospital″. The experimental group (EG) was formed of 12 patients (100.0 %) with LC of different etiology (6 people (50.0 %) – toxic-alimentary, 1 (8.3 %) – C-viral, 2 (16.7 %) – primary biliary, 2 (16.7 %) – toxic-alimentary + C-viral, 1 (8.3 %) – autoimmune + toxic-alimentary. The control group (CG) was formed of 10 practically healthy persons of the corresponding age and gender. Patients were provided with a comprehensive clinical-laboratory and instrumental examination of all organs and systems in accordance with the requirements of modern medicine. In addition, in all of the examined persons 10.0 ml of urine morning portion was taken, it was prepared according to the procedure, and subsequently the proteins with acetone were precipitated in a ratio of 1:6 and subjected to SDS-electrophoresis followed by Western blot analysis using commercial monospecific anti-cortactin antibodies. Statistical processing of information was carried out using programs Microsoft Office Excel 2010 with the estimation of the authenticity of the revealed changes using the Student’s t-criterion (W. S. Gosett) (the difference was considered significant for p < 0.05) were done. Diagnostic sensitivity of the certain protein was determined by the diagnostic specificity, prognostic significance of positive results, prognostic significance of negative results, and diagnostic effectiveness of the parameter. Results. During the study of the spectrum of immunologically reactive proteins in urine specimens of 12 patients with LC it was found that 9 of 12 patients had polypeptide with a molecular weight of 64 kDa, 4 subjects – 52 kDa protein, 8 – 24 kDa protein. It also has been found that immunoglobulin-related cortactin polypeptides with a molecular weight of 98, 64 and 24 kDa were present in the CG – 9 out of 10 urine specimens of healthy people contained protein with molecular weight of 64 kDa, one – 24 kDa, one – 98 kDa. According to the comparative analysis of the results obtained, it can be argued that 24 kDa protein found in 8 patients with LC was observed only in one of 10 urine specimens of the controls; 64 kDa polypeptide – in 9 persons of EG and 9 controls; 98 kDa – in nobody from the EG and only in the urine of one patient of the CG; 52 kDa protein – in 4 persons of EG and in nobody from the controls. 52 kDa protein, found in individuals of the EG, was not detected in the urine of any patient from the CG. After analyzing the information on 4 patients in whose urine 52 kDa protein was identified, it was found that in all LC was at the stage of decompensation (class C according to the criteria of C. H. G. Child – R. N. Pugh), in connection with which it can be assumed that the appearance of this protein in urine may be associated with the LC severity increase. 24 kDa protein was significantly more commonly found in the EG (8 persons – 6 patients had LC at the stage of decompensation, class C according to the criteria of C. H. G. Child – R. N. Pugh, in two – at the stage of subcompensation, class B according to the criteria of C. G. Child – R. N. Pugh) than in the CG (1 person) (p = 0.0039). Conclusions. Investigation of protein content in urine using western blot analysis using commercial antibodies to human cortactin allowed the identification of various molecular forms of peptides that differ in patients with liver cirrhosis of different etiology and healthy persons: the protein with the molecular weight of 52 kDa was present only in the urine of the experimental group patients, 24 kDa polopeptide was significantly (p = 0.0039) more likely common in the experimental than in the control group. The increase of the detection rate of 24.0 kDa proteins and the appearance of 52.0 kDa proteins in the urine may be associated with the appearance of the decompensated stage of liver cirrhosis. However, the assessment of their diagnostic value needs the further research.

Keywords

• cortactin
• diagnostics
• electrophoresis
• liver cirrhosis
• western-blot analysis