Effect of vitamin D and B12 levels on hepatosteatosis in overweight and obese children

Background. The childhood non-alcoholic hepatosteatosis leads to significant morbidity and mortality in adulthood. In recent years, vitamin D and B12 deficiency has been controversially associated with non-alcoholic fatty liver disease. Therefore, we aimed to evaluate the relationship of vitamin levels and insulin resistance (IR) with non-alcoholic hepatosteatosis in obese and overweight children. Material and methods. A total of 167 overweight and obese children aged 5–18 years were enrolled in this prospective study. The anthropometric measurements including body weight, height, body mass index (BMI) (weight/height2, kg/m2) were recorded. Children and adolescents with ≥ 95th percentile of BMI for their age and gender were diagnosed with obesity, and BMI between the 85th and 94th percentiles was classified as overweight. Children and adolescents classified into two groups according to the presence of hepatosteatosis: normal liver and hepatosteatosis group. Additionally, hepatosteatosis grading was performed by ultrasonographic evaluation. Participants’ demographic characteristics, physical examination, imaging and laboratory findings including serum levels of vitamin B12, 25(OH)D analysis and insulin resistance index were documented and compared between groups. Results. One hundred and sixty-seven patients included in this study were: 103 (61.7 %) males, 64 (38.3 %) females, and the mean age of total participants was 11.48 ± 2.99 years.  According  to  BMI-Z  score,  26.3  %  of  individuals  were  defined  as  overweight  and  73.7  %  were  obese. Hepatosteatosis was determined in 70.7 % (n = 118) of our cases and it was significantly higher in male patients (79.6 %) than in females (p = 0.001). Mean IR was statistically higher in non-alcoholic fatty liver disease group (11.15 ± 13.39) than in normal liver group (6.95 ± 6.20) (p = 0.029). Moreover, there were statistically significant differences found in IR levels according to severity of hepatosteatosis (p = 0.013). In addition, vitamin D and B12 deficiencies were not significantly associated with hepatosteatosis or severity of hepatosteatosis (p > 0.05). Also, no statistically significant differences were found in mean levels of vitamin D, vitamin B12 and IR between obese and overweight children. Conclusions. Our findings support the published data that vitamin D and B12 deficiencies do not contribute to the pathology of hepatosteatosis. In addition, insulin resistance has been demonstrated to be a risk factor for non-alcoholic fatty liver and hepatosteatosis severity.


Introduction
The Centers for Disease Control and Prevention (CDC) reported (2015) a 2-fold increase in prevalence of childhood obesity and a 4-fold increase in adolescent obesity within the last 30 years [1]. Nonalcoholic fatty liver disease (NAFLD) is the most frequently reported chronic pediatric liver disease in children and adolescents. NAFLD preva-lence appears to be increasing parallel to childhood obesity in recent years and it was reported in approximately 60 % of overweight children [2].
Although the pathophysiological mechanism of hepatocellular injury remains unclear, insulin resistance (IR) and metabolic syndrome are documented as risk factors in NAFLD. Long-term exposure to childhood-Mìžnarodnij endokrinologìčnij žurnal,ISSN 2224-0721 (print), ISSN 2307-1427 (online) Оригінальні дослідження /Original Researches/ onset NAFLD is associated with significantly increased morbidity and mortality in adulthood due to severe hepatocellular damage and fibrosis [3]. Such that childhood NAFLD has been strongly associated with cirrhosis, hepatocellular carcinoma, end-stage liver disease and cardiovascular diseases in adults [4]. Although USG and serologic screening based on liver transaminases (AST, ALT, GGT) and inflammation markers (CRP) are frequently used, liver biopsy is the gold standard method in the diagnosis of hepatosteatosis which is often asymptomatic in childhood [3].
Vitamins D and B 12 deficiency or insufficiency have been significantly associated with obesity, overweight, type 2 diabetes mellitus, metabolic syndrome, IR and cardiovascular disease in recent years [5][6][7]. It is well known that immunomodulatory and anti-inflammatory characteristics of vitamin D inhibits hepatic inflammation and fibrosis [8]. Moreover, low serum concentrations of 25-hydroxyvitamin D have been significantly associated with NAFLD and with or without non-alcoholic steatohepatitis (NASH) [9]. Similarly, vitamin B 12 deficiency triggered hyperhomocysteinemia is significantly associated with NAFLD [10]. Furthermore, even the severity of hepatosteatosis was associated with vitamin D and B 12 deficiency levels [10,11]. On the contrary, there are published data concluded that vitamin deficiencies play no role in the etiopathology of hepatosteatosis [12]. Thus, the findings related to the vitamin deficiencies and hepatosteatosis seems to be controversial.
The purpose was to evaluate the relationship between vitamin levels and IR with non-alcoholic hepatosteatosis in obese and overweight children. A total number of 167 overweight and obese children aged between 5-18 years old, were enrolled in present study. Children were enrolled in the study after obtainment of written informed consent from their family. The anthropometric measurements including body weight, height, BMI (weight/height 2 , kg/m 2 ) were recorded. Body weight, height and BMI percentile were determined by using the growth curves of Turkish children [13]. Children and adolescents with a ≥ 95 th percentile of BMI for their age and gender were diagnosed with obesity, and BMIs between the 85 th to 94 th percentiles were classified as overweight.

Materials and methods
Children and adolescents classified into two groups according to the presence of hepatosteatosis; absent hepatosteatosis group and hepatosteatosis group. Hepatosteatosis assessment performed by liver ultrasonography (Acuson S2000 Ultrasound system, Siemens, Germany). Additionally, hepatosteatosis grading was performed as follows: Grade 0: Normal parenchymal liver echotexture. Grade 1: Slightly increased liver echogenicity with normal visualization of vessel walls. Grade 2: Moderately increased liver echotexture with impaired visualization of the vessel walls.
Grade 3: Severe increased liver echogenicity with poor visualization of the liver, portal vein borders and the diaphragm [14].
The serum levels of vitamin B 12 and 25(OH)D analysis were performed using an immunodiagnostic system (Siemens, Advia Centaur xp, Germany) at a normality level of 220 pg/ml and 29.0 ng/ml, respectively. Biochemical analysis performed from serum samples by electro-chemiluminescence immunoassay analyzer (Beckman Coulter Unicel DXI 800). Haematological parameters were analysed using a haematology analyser (Cell-Dyne 3700, Abbott, Abbott Park, IL, USA). The insulin resistance index calculated according to the homeostasis model assessment of insulin resistance (HOMA-IR) formula; HOMA-IR = fasting serum insulin (microunits per milliliter) × fasting glucose (millimoles per liter)/22.5 [15]. Participants' demographic characteristics, physical examination, imaging and laboratory findings were documented and compared between groups.
Statistical analysis. All the data were analyzed with SPSS (Statistical Package for the Social Sciences) software for Windows (v21.0; IBM, Armonk, NY, USA). Individual and aggregate data were summarized using descriptive statistics including mean, standart deviations, medians (minmax), frequency distributions and percentages. Normality of data distribution was verified by Kolmogorov-Smirnov test. Comparison of the variables with normal distribution was made with Student t test. The variables which were not normally distributed, the Mann Whitney and Kruskal Wallis tests were conducted to compare between groups. Evaluation of categorical variables was performed by Chi-Square test. P-values of < 0.05 were considered statistically significant.
Moreover, there were statistically significant differences found in IR levels according to severity of hepatosteatosis (p = 0.013). Mean IR was significantly increased particularly in grade 2 hepatosteatosis. In addition, vitamin D and B 12 deficiencies were not significantly associated with severity of hepatosteatosis (table 3). There were also no

Discussion
Despite the increasing prevalence of NAFLD in children and adolescents, there are estimated to be a large number of undiagnosed children due to the asymptomatic characteristics of the disease, the absence of standardized guidelines, invasive and expensive diagnostic procedures. Additionally, NAFLD primarily affects male gender [3]. J.A. Welsh et al. reported 3 times higher male prevalence in a study consisting of 14 918 children diagnosed with NAFLD in 20 years period [16]. V. Nobili et al. documented the male percentage of 64 % in 73 children with NAFLD [11]. Similarly, children included in our study were 61.7 % male and 38.3 % female. Furthermore, hepatosteatosis was significantly higher (79.6 %) in male children.
Vitamin D deficiency or insufficiency has been significantly associated with increased BMI scores in published data. In addition, it is well known that obesity increases the risk of NAFLD. Therefore, the relationship between hepatosteatosis and childhood vitamin deficiencies has become an increasingly popular field of study in recent years with controversial findings [17]. G. Targher et al. reported significantly lower levels of 25(OH)D in histopathologically confirmed NAFLD children (n = 60) than healthy (n = 60) controls [18]. Similarly, in a meta-analysis consisting of 59 studies and 13 524 patients (5896 NAFLD and 7628 controls), researchers documented significantly lower serum 25(OH)D levels in NAFLD patients. Additionally, they no ted a 1.26-fold (OR 1.26, 95% CI 1.15 to 1.38) increase in the prevalence of NAFLD associated with vitamin D deficiency. Thus, researchers concluded that vitamin D deficiency may play a role in the pathogenesis of NAFLD and NASH [19]. On the contrary, F. Dursun et al. has not significantly associated serum vitamin D deficiency or insufficiency with hepatosteatosis in 110 obese children [20]. Supportively, K. Katz et al. identified overweight and male gender as risk factors for hepatosteatosis, but not vitamin D deficiency in a study included 1630 adolescents [21]. In accordance with these data, vitamin D deficiency or insufficiency were not significantly associated with hepatosteatosis or severity of hepatosteatosis in present study.
Although decrease in serum B 12 levels has been documented in acute hepatitis, cirrhosis and hepatocellular carcinoma, the relationship between vitamin B deficiency and NAFLD currently is not clear. There is limited number of published data with debated results evaluating vitamin B deficiency in NAFLD-patients. In a study, M. Koplay [24]. Supportively in our study, vitamin B 12 deficiency or insufficiency were not significantly asso ciated with hepatosteatosis or severity of hepatosteatosis.
It is unclear whether insulin resistance is a cause or a consequence of NAFLD. But IR related-liver fat accumulation is frequently reported and in some studies IR increase is defined as NAFLD feature even in non-obese patients [25]. J.F. Fu et al. divided 861 obese children (aged between: 6-16 years) into 3 groups; 'fatty livernormal ALT', 'fatty liver -elevated ALT' and 'normal liver -normal ALT'. Researchers have revealed that IR was significantly increased in fatty liver groups [26]. Similarly, in a study conducted with 846 children, K. Tominaga et al. histopathologically confirmed the NAFLD diagnosis in 37 children and concluded that NAFLD was closely associated with metabolic syndrome and IR [27]. In a study conducted by A. Fraser et al. with 5586 adolescents, researchers demonstrated significantly increased serum ALT, CRP, triglyceride and IR levels in children with NAFLD [28]. In accordance with these data, mean serum levels of hemoglobin, ferritin, AST, ALT, GGT, triglyceride were statistically higher and mean HDL was statistically lower in NAFLD group than normal liver group in our study. Additionally, mean IR was statistically higher in NAFLD group than normal liver group. Moreover, it is remarkable that mean IR was significantly increased particularly in grade 2 hepatosteatosis.

Conclusions
Our findings support the published data that vitamin D and B 12 deficiencies do not contribute to the pathology of hepatosteatosis. It should be considered that vitamin deficiency may occur particularly in obese pediatric population due to the already existing hepatocellular damage and fibrosis. In addition, insulin resistance has been demonstrated to be a risk factor for non-alcoholic fatty liver and hepatosteatosis severity.

Conflicts of interests.
Authors declare the absence of any conflicts of interests and their own financial interest that might be construed to influence the results or interpretation of their manuscript.
Funding sources: none.