MORPHOFUNCTIONAL CHANGES OF GALLBLADDER AND BIOCHEMICAL PARAMETERS OF LIPID METABOLISM IN CHILDREN WITH LIVER STEATOSIS

. Background. The aim of study was to investigate peculiarities of structural and functional disorders of gallbladder in children with non-alcoholic fatty liver disease. Materials and methods. The study was conducted in 51 children aged 10–17 years. Patients were divided into four groups: the 1 st group consisted of 11 children with biliary normokinesia without steatosis; the 2 nd group — 20 children with biliary hypokinesia without steatosis; 3 rd group — 11 children with biliary normokinesia and steatosis, and the 4 th group — 9 children with biliary hypokinesia and steatosis. Anthropometric parameters were measured: height, weight, abdominal circumference, body mass index. Functional state of gallbladder was investigated with the help of ultrasound. Such biochemical parameters, as total cholesterol, triacylglycerols, levels of high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and very-low-density lipoprotein cholesterol were defined, atherogenic index was calculated according to Friedewald formula. Results. Present study shows overweight (in 15 % of patients), obesity (in 85 % of patients), increase in the gallbladder volume by 30–50 % (p < 0.05), in the density of gallbladder wall by 12–16 % (p < 0.05) and its echogenicity by 24 % (p < 0.05) in children with steatosis versus group without steatosis and gallbladder normokinesia. Positive correlation between wall thickness and steatosis has been established. Thus, weight increase may potentially play a role in lipid disorders and biliary dysfunction. The 70 % of children with obesity had pathological changes of lipid levels, such as 74 % — increased blood atherogenicity. Conclusions. Physicians must necessarily correct a functional state of gallbladder during the treatment of obesity in children and adolescents. Only comprehensive approach to the treatment allows prevention of non-alcoholic fatty liver disease and its complications.


Introduction
Since 1980, number of patients with obesity increased twice. In 2014 more than 41 million of children older than 5 years had overweight. Physicians set obesity apart as a systemic disease which is based on lipid and carbohydrate metabolism disorders [1]. In blood of patients with overweight level of free fatty acids is increased. It leads to fat accumulation in internal organs and visceral obesity. Therefore, overweight is a main etiological factor in the pathogenesis of metabolic disorders, such as nonalcoholic fatty liver disease (NAFLD) and functional gallbladder disorders (FGD) [2][3][4]. In Calasani's study (2012) among adult patients with NAFLD 90% had in-creased body mass index (BMI) and visceral obesity, in 69 % of cases diabetes mellitus type 2 was diagnosed, in 50 % of cases -high level triacyl glycerides (TG) and low level of high density lipoprotein (HDL) in blood serum were found [5]. Such changes are common for pediatric patients. Clinical investigation showed NAFLD in one third of children with obesity, which was often accompanied by dyslipidemia, intolerance to carbohydrates and insulin resistance [6]. Etiology, development and diagnosis of steatosis in children are specific. The youngest age of steatosis diagnosis is 2 years [7]. Ecological and genetic factors play important role in NAFLD development [5]. Our previous investigations demonstrated that more than 50 % of children with NAFLD had FGD [8]. The previous studies showed positive correlation between total obesity, fatty liver and fatty gallbladder in adults. Under these conditions the functional state of liver and gallbladder is low. The existing literature data describe a direct link between the overall adult obesity, fatty liver and fatty gallbladder, thus decreasing their functional ability. Unfortunately to date there is no detailed analysis of the relationship of these abnormalities in pediatric patients.
Aim of study -to investigate peculiarities of structural and functional disorders of gallbladder among children with non-alcoholic fatty liver disease.

Materials and methods
Study was conducted in children and adolescents searching for medical help with gastro-intestinal tract disorders in SD «Institute of Gastroenterology of the National Academy of Medicine Sciences of Ukraine». All patients had given their agreement to participation in the study.
The study was conducted in 51 children aged 10-17 years. Patients were divided into four groups: the 1 st group consisted of 11 children with biliary normokinesia wi thout steatosis; the 2 nd group -20 children with biliary hypokinesia without steatosis; 3 rd group -11 children with biliary normokinesia and steatosis, and the 4 th group -9 children with biliary hypokinesia and steatosis.
Anthropometric parameters were measured: height, weight, waist circumference, body mass index:

BMI = [body mass, kg]/[height, m] 2 [9].
Thickness of gallbladder wall and its volume were measured in all patients with ultrasound (SН-2000 Нonda Electronics) in real time scale, on an empty stomach. Analysis of changes in abdominal organs included size measurement, profile, acoustic structure and echogenicity by generally accepted methods [10].
We had studied gallbladder function with ultrasound on the background of choleretic breakfast. As choleretic breakfast we used more physiological routine food loading: 40 g of wheat bread, 20-25 g of butter, 150-200 ml black tea with 5 g of sugar [11].
Gallbladder volume was measured: with empty stomach and after breakfast (5, 10, 45 and 60 th minutes).
Decreasing of gallbladder volume on 45 th or 60 th minutes after breakfast for 34-64 % was the evidence of gallbladder normokinesia, less than 33 % -hypokinesia [11].
Diagnosis of liver steatosis was realized by measurement of the controlled attenuation parameter (CAP) with FibroScan ® 502 Touch F60156 (Echosens, France). This parameter was correlated with the degree of fatty liver. Its level higher than 232 db/m 2 confirmed that total amount of fatty hepatocytes was more than 10 %.

Table 1. Anthropometric and sonographic parameters in children depending on functional state of gallbladder and presence of steatosis
Data from all groups were compared using the Student unpaired t-test by SPSS 9.0 for Windows. Difference average values were considered as probable in р < 0,05 and р < 0,01 [13].

Results
Study showed no difference in patients age and height between groups (table 1), but body weight was significantly increased in groups 3 rd and 4 th on average 45 % in comparison with 1 st group.
High body weight led to BMI increasing by 26 % in children with steatosis in comparison with children without steatosis. Abdominal type of obesity was presented in children with overweight. In 3 rd group waist circumference increasing in 1,8 times was observed, subcutaneous fat increasingby 59 % and intraperitoneal fat increasingby 10-57 %. So, patients from groups 3 rd and 4 th were characterized by overweight (15 % of cases) and obesity (85 % of cases) (p  0,05). In 1 st and 2 nd groups obesity was observed in 43 and 24 % respectively.
Decreasing of gallbladder motility directly influenced on digestion and absorption of lipids in the intestine, such as total lipid metabolism in organism [2]. About 70 % of children with obesity had a pathological changes of lipid levels, such as 74 %increasing of blood atherogenicity.
In our study hypercholesterinemia was observed in every group, its frequency was 50 % higher among patients with gallbladder hypokinesia: 60 % patients in 1 st group and 29 % patients in 4 th respectively ( fig. 1).
Thus, hypertriacylglycerolemia prevalenced among patients with steatosis ( fig. 2). It could be related to high triacylglycerides production in the liver as a main mechanism of steatosis development.
Analysis of the lipid fraction ratio in the blood serum depending on gallbladder functional state and liver steatosis in children showed changes in TG, PL, HDL, LDL, VLDL, CH/PL levels and AC. The mild atherogenic dyslipidemia was found in the 3 rd group. There were increasing in 1,4 times TG level (р < 0,05), VLDL -in 1,5 times (р < 0,05) and increasing of CH/PL rate in 1,3 times (р < 0,05), in comparison with 1 st group (table 3).

Discussion
Low gallbladder motility with increased volume and wall thickness were found in children with steatosis. This disorder was accompanied by atherogenic changes in lipid fractions (high TG and VLDL levels). They were more severe in groups with steatosis. So, obesity and overweigh are underlying causes in NAFLD development, and FGD leads to the complication of disease. It also does not exclude the deterioration functional status of the gallbladder due to general obesity.
Our data demonstrated an increasing of gallbladder volume and wall thickness in children with steatosis. Significant increase of these parameters in III and IV groups in comparison with I group was showed. Also, motility of gallbladder in these groups was less than in children without steatosis. Correlation between NAFLD and FGD can be explained by lipotoxicity effect which leads to lipid accumulation outside of adipose tissue. Free fatty acids and cholesterol play the key role in this mechanism [14]. It is assumed, that cholesterol increase and relation of cholesterol to phospholipids is a cause of low gallbladder motility because of humoral response inhibition by neuronal transmitters such as acetylcholine, cholecystokinin and neuronal peptide Y [15-17]. It was evidenced in the experiments with animals. Blocking of intestinal absorption of cholesterol led to decrease of lipids level in gallbladder wall and improved gallbladder motility [18].
Other eventual mechanism of low gallbladder motility in NAFLD is an effect of hyperproduction of cytokines in adipocytes which leads to visceral organ inflammation.
Numerous studies showed the fact that adipocytes is capable to produce inflammatory molecules, including TNF-, IL-6 and cause inflammation in other organs [19][20][21].
So, inflammation in patients with overweight and obesity cause weak function of gallbladder wall and decrease absorption and secretion ability [22,23]. Our results proved this conception because patients with NAFLD in our research groups often had a high BMI and enlarged gallbladder volume.
Overweight is one of the causes of gallbladder hypokinesia. We suggest that two mechanisms may play role in development of gallbladder hypokinesia in steatosis. They are based on accumulation of fat in gallbladder wall and decreasing of humoral response to neurotransmitters action as a result of cholesterol/phospholipids relation disruption and inflammation. In our opinion, physicians must necessarily correct a functional state of gallbladder during treatment of obesity in children and adolescent. Only complex approach of treatment allows prevention of non-alcoholic fatty liver disease and its complications [24].

Conclusions
1. Overweight and abdominal type of obesity play key role in the development of fatty liver in children.
2. Decreased gallbladder contractility lead to dislipidemia, such as hypercholesterinemia development whereas steatosis is associated with hypertriacylglycerolemia.