Diagnostic significance of biochemical inDicators of liver fibrogenesis in aDolescents with obesity

Non-alcoholic fatty liver disease occurs in most obese people, the main pathway of which is the process of fibrogenesis. The aim of this work was to determine the potential biomarkers for early diagnosis of liver fibrogenesis in adolescents with obesity. The levels of liver fibrosis markers, such as fibronectin, collagen type IV, N-terminal propeptides and C-terminal telopeptides of type I collagen, were assessed with the use of IFA method in serum of 226 patients with obesity aged 8-18 years. A significant increase in levels of type IV collagen and fibronectin was observed in children with obesity (P < 0.05). As diagnostic criteria for fibrogenesis and fibrolysis, the levels of N-terminal propeptides and C-terminal telopeptides of type I collagen, respectively, were determined. The serum level of N-terminal propeptides of type I collagen significantly exceeds the normal values in all children with obesity compared to the control group (P < 0.05). The biochemical markers (type IV collagen, fibronectin, N-terminal propeptides and C-terminal telopeptides of type I collagen) were proven to have high diagnostic informative value in the early diagnosis of liver fibrogenesis in obese adolescents. It was shown that the signs of fibrosis in non-alcoholic fatty liver disease already occur at the stage of steatosis.


O. V. BUzNytSka
Kharkіv Medical Academy of Postgraduate Education, V. N. Karazin Kharkіv National University, Ukraine e-mail: ebuznickaa@ukr.net;missbuzelena@gmail.comreceived: 27 September 2018; accepted: 13 December 2018 Non-alcoholic fatty liver disease occurs in most obese people, the main pathway of which is the process of fibrogenesis.The aim of this work was to determine the potential biomarkers for early diagnosis of liver fibrogenesis in adolescents with obesity.The levels of liver fibrosis markers, such as fibronectin, collagen type IV, N-terminal propeptides and C-terminal telopeptides of type I collagen, were assessed with the use of IFA method in serum of 226 patients with obesity aged 8-18 years.A significant increase in levels of type IV collagen and fibronectin was observed in children with obesity (P < 0.

05). As diagnostic criteria for fibrogenesis and fibrolysis, the levels of N-terminal propeptides and C-terminal telopeptides of type I collagen, respectively, were determined. The serum level of N-terminal propeptides of type I collagen significantly exceeds the normal values in all children with obesity compared to the control group (P < 0.05). The biochemical markers (type IV collagen, fibronectin, N-terminal propeptides and C-terminal telopeptides of type I collagen) were proven to have high diagnostic informative value in the early diagnosis of liver fibrogenesis in obese adolescents. It was shown that the signs of fibrosis in non-alcoholic fatty liver disease already occur at the stage of steatosis. K e y w o r d s: adolescents, non-alcoholic fatty liver disease, liver fibrosis, obesity, diagnostics methods.
I n modern hepatology, many factors that contribu tetothedevelopmentofchronicdiffuse liver disease are known.These include viruses, alcohol, drug compounds, toxins, genetically caused metabolic disorders.Currently, non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in the world, which combines the range of clinical and morphological changes representedbysteatosis,steatohepatitis,steatofibrosis and liver cirrhosis [1][2][3][4].The modern model of the pathogenesis of NAFLD is represented by the theory of"twostrokes",thefirstofwhichisthedevelopment of fatty degeneration, the second one is steatohepatitis.Upon obesity, especially abdominal, and in conditions of insulin resistance (IR), the lipolysis of adipose tissue increases leading to an increase in the supply of free fatty acids to the liver and the formationofsteatosis.Thisisseenasthe"firstblow".
Prolonged increase in the level of free fatty acids in hepatocytes causes mitochondrial dysfunction, which contributes to the microsomal oxidation of lipids by the cytochrome P450 system with the formation of reactive oxygen species [5][6][7].The oxidative stress, the "second blow", occurs.That results in increasedformationofproinflammatorycytokines withtheformationofinflammatoryreactionsinthe liver and the development of steatohepatitis.At certainstagesofinflammation,cellularinfiltrationand dystrophy are joined by the activation of liver stem cells, which begin to synthesize the components of the intercellular matrix in surplus quantities (collagen of various types, laminin, fibronectin, and others ).Theonsetoffibrogenesisisaccompanied,in mostcases,bythedecreaseofsignsofinflammation andthepredominanceoffibrosis,whichleadstoa violation of the organ structure with the subsequent development of liver cirrhosis and even hepatocellular carcinoma [8,9].
One of the components of the intercellular spaceisfibronectin.Thisglycoproteinisa"molecularglue"thatpromotesthefixationofcellsinthe intercellular space by interacting with membrane receptors, and also enhances phagocytosis [10].Withthedevelopmentoffibrosis,boththequantitative and qualitative composition of the intercellular matrix vary, in particular, the level of collagen of differenttypesincreases.Oneofthefirstinvarious liver lesions is collagen type IV, which is the main structural element of cellular basement membranes.ThelevelofcollagentypeIVreflectscurrenthepatocellular damage in the initial stages and processes of liver regeneration [11,12].In the future, the accumulation of other types of collagen, in particular type I, leadstotheformationofbridge-likefibrosisandultimately contributes to the development of cirrhosis.N-terminal propeptides and C-terminal telopeptides of type I collagen are used as diagnostic criteria for fibrosisandfibrolyses,respectively [13].
Theactualincidenceofthediseaseisdifficult to establish, due to the inadequate use of biochemical screening diagnostic methods, through which the initial forms of the disease can be detected [14][15][16].To date, the most informative and objective method of diagnosis of NAFLD remains liver biopsy, which provides the possibility to assess the stage, exclude other causes of liver damage and predict further course of the disease [17].But due to significant restrictions, namely: the acquisition of informative material, the risk of complications, childhood, the complexity of further dynamic observation, there are grounds for the development of non-invasive, safer and more sensitive diagnostic methods, mainly biochemical and molecular, which highly correlate with the parameters of liver biopsy and easly available in clinical practice [18].Therefore, in recent years, interest in the search for biochemical markers of liverfibrogenesis [19]hasbecomeconsiderablyaggravated, which in the future would contribute to the development of algorithms for primary prevention and early diagnosis of NAFLD.
The aim of this work was to study the diagnosticsignificanceofbiochemicalmarkersofliver fibrogenesisinadolescentswithobesity.

material and methods
The research was performed at the endocrinology department of SI "Institute of children and adolescence health care of NAMS" (Kharkov).226 children with obesity aged 8 to 18 years were exami ned: 129 males (57.08 ± 3.29%), 97 females (42.92 ± 3.29%).The control group consisted of 30 healthy children of the same age group.To assess the functional status of the liver in adolescents with obesity, a complex of clinical, laboratory, biochemical and instrumental research methods was used.The criterionforIRwasthehomeostaticmodelНОМА-IR (Homeostasis model assessment of Insulin Resistance, Matthews D.R., 1985).The calculation was made using formula: HOMA = (G0•Ins0)/22.5;(G0fastingglucose level of the blood serum, mmol/l; Ins0-contentofinsulininbloodserum,μU/ml.The Resultismorethan3.5unitstestifiedtothepresence of IR. Immunoassay analysis was used to determine thebiochemicalmarkersoftheintensityofliverfibrogenesis,namelyfibronectininplasma(Fibronectin ELISA Kit, Company Biohimmak (RF)), serum type IV collagen (Serum collagen IV EIA, Argutus Medical (Japan)) in blood, N -terminal propeptides of type I collagen (NT-proCNP, Biomedica (Austria)) and C-terminal telopeptides of type I collagen (Serum CrossLaps Elisa, Immunodiagnostic Systems Ltd (UK)).Blood collection and stabilization were carried out according to the instructions for the kits.The reference maximum value for collagen type IVis99.0±2.3μg/linserum;whilefibronectinis70.0±14.0μg/mlinbloodplasma.Theconcentration of N-terminal propeptides and C-terminal telopepep tides of type I collagen in serum varies depending on age and sex.
Database creation and statistical processing of the results were performed on IBM-Pentium III using application packages "Stadia-6" (serial num-beroflicensecertificate1218May24,2000,version "Prof"), Microsoft "Access", "Excel".The t-criterion of the Student (p) was used to assess the likelihood ofdifferences,aswellascorrelationanalysis.The critical significance level for checking statistical hypotheses when comparing groups was assumed to be 0.05.Ethical norms at all stages of the survey were observed.The work was conducted taking into account the requirements of the European Convention (Strasbourg, 1986), the provisions of the ICH GCP (2008), GLP (2002).Studies did not cause psychological discomfort in patients.Patients and their parents were provided with information on the methods and scope of the research, signed informed consent to participate in the study.

results and Discussion
IRwasidentifiedin113of226(50.0%)patients, according to the results of the index NOMA-IR.
Individual analysis of patients in groups was made.It showed these indicators in obese patients, maximumlevelsfortypeIVcollagen:232.11μg/linateenagerwithIR;198.11μg/l-withoutIRandfibronectin:253.2μg/mlinpatientwithIR;200.0μg/ml-withoutIR.Itshouldbenotedthatitwasin these patients that according to ultrasound examination of the liver, were the most distinct changes: a sharp increase in the liver, a decrease in echogenicity, seizure of the walls of the vessels, indicating unfavorable dynamics of the disease.Using correlation analysis, a statistically valid direct relationship of ave rage strength was found between the levels of bio chemical markers and ultrasound results (r = +0.4;Р < 0.05).
As the diagnostic criteria of the two physiologicallyantagonisticprocesses-fbrogenicandfbrosis, we determined the levels of N-terminal propeptides and C-terminal telopeptides of type I collagen, respectively, with subsequent assessment of the benefitsofoneofthem.
The results obtained for N-terminal propeptides of type I collagen in serum are presented in Table 2, whichshowsthatitslevelsignificantlyexceedsthe reference values in all obese adolescents, unlike children in the control group (P < 0.05).
It should be noted that in teenagers with IR, the levels of N-terminal propeptides type I collagen were more elevated than in the non-IR group, whichindicatesanintensiveprocessofliverfibrosis in presence of IR (P < 0.05).In the prepubertalperiod,thisindicatorwassignificantlyhigherin girls with IR (11.12 ± 1.6), unlike boys (9.03 ± 0.21) (P < 0.05), and in the postpubertal period, on the contrary -boys with IR had significantly higher values of N-terminal propeptides (8.28 ± 0.8) than girls (5.51 ± 0.88) (P < 0.05).It is connected with the fact that in the prepubertal and puberty period, the growth spurt of girls is physiologically faster than that of boys who "catch up" with girls in the middle of puberty and postpuberty periods.
Thus, the exchange of connective tissue, its regeneration is most active during intense growth.In the group of children without IR, the level of biochemical markers in pubertal and postpubertal adolescent boys (9.536 ± 1.84 and 7.806 ± 0.94 respectively)significantlyexceededtheratesamong girls of the same age (6.903 ± 0.61 and 4.536 ± 0.52, respectively) (P < 0.05).That is, it can be argued that thereareearlystagesofliverfibrosisinadolescents with obesity, which are more prominent in those surveyed with IR.The levels of the studied index in the control group were within the normal range (P < 0.05).

T a b l e 1. Levels of type IV collagen and fibronectin in adolescents with obesity (M ± σ)
The results of C-terminal telopeptides of type I collagen in serum are presented in Table 3.
As can be seen from the data obtained, the levels of C-terminal telopeptides of type I collagen in obese adolescents were within the reference norms and did not differ statistically significantly from such control groups (P > 0.05).Exceptions are the children of puberty age, both with IR (1.88 ± 0.18 boys and 1.673 ± 0.18 girls) and without it(1.888±0.34boysand1.281±0.10girls),whosefibrolysismarkerindicesweresignificantlylowerthan in control group of children (2.61 ± 0.71 males and 2.47 ± 0.68 girls) (P < 0.05).Most likely, this is due tothepredominanceoffibrogenesisoverfibrolysis processes,whichischaracteristicofliverfibrosis.Therewasalsoasignificantprevalenceofthisindicator in girls of postpubertal age with IR, as opposed to girls without IR (1.134 ± 0.14 and 0.733 ± 0.06 respectively, P < 0.05).
According to the results of the correlation analy sis, in adolescents with IR a direct correlation of average strength between the level of N-terminal propeptides and C-terminal telopeptides of type I collagen (r = 0.461; P < 0.01) was established.Thus, with the predominance of fibrogenesis processes, thesystemoffibrolysisisautomaticallyactivatedto maintain the physiological balance of the connective tissue metabolism.In patients without IR, a direct correlation of average strength between the content of propeptides and telopeptides of type I collagen (r = 0.452; P < 0.001) was also established; and so was a strong correlation between the levels of col-lagen type IV and C-terminal telopetides (r = 0.709; P < 0.05) and average strength correlation with the level of N-terminal propeptides of type I collagen (r = 0.467; P < 0.05).
Thus, the obtained data indicate the presence offibrogenesisprocessesevenattheinitialstages of fatty liver disease in adolescents with obesity.Theproofisthatthereisasignificantincreasein levelsofcollagentypeIVandfibronectin,incontrast to the control group (P<0.05).Indicatorsoffibronectinsignificantlydifferedingroupsdepending on the presence of IR, which is probably associated with more severe liver damage in children with IR (P < 0.05).The established changes in N-terminal propeptides and C-terminal telopeptides of type I collagenindicatethepredominanceoffibrogenesis processesoverfibrolysisprocesses,whichalsoindicatethepresenceoftheearlystagesofliverfibrosis, long before the clinical manifestation of NAFLD and confirmationbyavailablediagnosticmethods.