Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Hepatology and Nutrition
Serum Retinol-binding Protein 4 Is Independently Associated With Pediatric NAFLD and Fasting Triglyceride Level
Huang, Shu-Ching*; Yang, Yao-Jong†
*Department of Pediatrics, Kuo General Hospital
†Department of Pediatrics, National Cheng Kung University Hospital, Medical College, National Cheng Kung University, Tainan, Taiwan.
Address correspondence and reprint requests to Yao-Jong Yang, MD, PhD, Department of Pediatrics, National Cheng Kung University and Hospital, #138, Sheng Li Road, Tainan 704, Taiwan (e-mail: firstname.lastname@example.org).
Received 17 January, 2012
Accepted 31 August, 2012
The study was supported by grant KGH-9825 from Kuo General Hospital, Tainan, Taiwan, and DOH098-TD-F-113–098031–2 from the Department of Health, Taiwan.
The authors report no conflicts of interest.
Objectives: Nonalcoholic fatty liver disease (NAFLD) is identified as a major liver disease in children. The present study aimed to identify the prevalence and predictors of pediatric NAFLD and the correlation between serum retinol-binding protein 4 (RBP4) levels and metabolic characteristics in children.
Methods: A total of 748 schoolchildren, ages 6 to 12 years, were enrolled in 2009. The body weight and height were measured in the morning before intake. Laboratory tests included overnight fasting serum lipids, insulin, liver enzymes, and RBP4 levels. Hepatic steatosis was determined by ultrasound in 219 volunteers.
Results: The rates of NAFLD were 3% in the normal-weight, 25% in the overweight, and 76% in the obese children. Twenty (22%) of obese children had abnormal alanine aminotransferase (ALT) levels. In children with NAFLD, younger age and higher body mass index (BMI), insulin/homeostasis model of assessment, and male sex rate were associated with abnormal liver function. Stepwise increments in BMI, insulin, homeostasis model of assessment, and ALT were found in children with normal livers to simple steatosis, and to steatosis with abnormal ALT. Multiple logistic regression analysis confirmed that serum RBP4 levels (P = 0.048), ALT (P = 0.048), and BMI (P < 0.001) were independently predictors of pediatric NAFLD. Moreover, multiple linear regression analysis revealed that only serum triglycerides levels were positively related to RBP4 levels (P < 0.001).
Conclusions: Higher RBP4 and ALT levels as well as BMI are independently associated with pediatric NAFLD in Taiwan. In addition, an increment in RBP4 levels was positively correlated to hypertriglyceridemia in children.
Nonalcoholic fatty liver disease (NAFLD) has been increasingly recognized worldwide in the last decade (1–5). There have been studies reporting that the prevalence of NAFLD in the general population is 2.6% but increases to 60% in obese children (3,4). Consequently, NAFLD is anticipated to have catastrophic future influences. NAFLD encompasses a broad spectrum of liver abnormalities ranging from simple steatosis to nonalcoholic steatohepatitis and cirrhosis (6,7). At present, NAFLD is identified as a major liver disease in children, occurring even in the extremely young (8).
Obesity, hyperglycemia, type 2 diabetes mellitus, and hypertriglyceridemia are risk factors for the development of NAFLD (9–11). NAFLD is hence regarded as a manifestation of metabolic syndrome. Insulin resistance has been shown to play a key role in the pathogenesis of NAFLD in obese adults and adolescents (12,13). Considering that obesity is the most significant predisposing factor for insulin resistance, adipokines have become the focus of research for NAFLD (14,15). Retinol-binding protein 4 (RBP4), secreted mainly from the liver and adipose tissue, has recently been proposed to be a link between obesity and insulin resistance (16). Moreover, insulin resistance as well as metabolic syndrome often begins in childhood or young adulthood (17). Therefore, it is particularly important to investigate the role of RBP4 on childhood obesity and NAFLD.
An increment in RBP4 concentrations is correlated with increased adiposity in children (18,19). Moreover, RBP4 levels have been related to weight status and insulin resistance in both cross-sectional and longitudinal analyses (19). It has been proposed that RBP4 may be a mechanism through which obesity influences insulin resistance and hypertriglyceridemia in overweight youths (18). Therefore, the utility of RBP4 as a biomarker of insulin resistance in children at risk has been suggested. A negative association between serum RBP4 levels and hepatocellular injury has been found in children and adults with histopathology-proven NAFLD (20,21). Because NAFLD is an alarming clinical feature leading to the development of advanced liver disease in obese children, the present study aimed to identify independent risk factors for pediatric NAFLD and to investigate the correlation between serum RBP4 levels and metabolic characteristics of NAFLD.
The participants in the present study, ages 6 to 12 years, were randomly recruited from 6 elementary schools in Tainan City and County, Taiwan, in 2009. Subjects were excluded if they had a history of alcohol drinking or drug consumption, formerly diagnosed liver disease, or diabetes. The details of the present study were well explained to participants and their parents, and written informed consent was acquired. This research was approved by the ethics committees of National Cheng Kung University Hospital (ER-98–050) and Kuo General Hospital (ER-09-K004).
Clinical and Laboratory Evaluation
Clinical data such as age, sex, height, and weight were recorded. Height was measured to the nearest 0.1 cm and weight to the nearest 100 g using a digital balance. Body mass index (BMI) was calculated as weight (kg)/height2 (m). Age- and sex-specific criteria from the nationwide standards of the Department of Health, Taiwan (22), were adopted to categorize the children as normal-weight, overweight (BMI between the 85th and 95th percentile), and obese (BMI >95th percentile).
Blood samples were drawn after an 8-hour overnight fast and immediately centrifuged. Serum triglycerides (TG), total cholesterol, high-density lipoprotein (HDL)-cholesterol, glucose, insulin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured instantly by automated methods. Insulin resistance was evaluated using the homeostasis model of assessment (HOMA), calculated as fasting glucose (mmol/L) × fasting insulin (mU/L)/22.5 (23). Hepatitis B surface antigen, anti-hepatitis C virus antibody, and serum ceruloplasmin (IMMAGE Immunochemistry System, Beckman Coulter, Brea, CA) levels were screened in the participants with elevated serum ALT values (>40 IU/L). Serum RBP4 concentrations were measured with an enzyme-linked immunosorbent assay using a commercial kit (Immundiagnostik AG, Bensheim, Germany). The intra- and interassay coefficients of variation were 5.0% and 9.8%, respectively.
To minimize the confounder factors taken into account for analysis, NAFLD was diagnosed on ultrasonography by a single experienced pediatric gastroenterologist who was unaware of the participants’ biochemical profiles. An ultrasound device with a 3.5-MHz convex-type transducer (Toshiba/SSA-550A, Tokyo, Japan) was used. Of the 4 known criteria used for the diagnosis of fatty liver (hepatorenal echo contrast, liver brightness, deep attenuation, and vascular blurring), the first 2 were used as the definitive criteria, and the last 2 were taken into account as needed (1). Participants with NAFLD were subsequently divided into 2 groups according to serum ALT activity. Subjects with steatosis in the ultrasound scan and normal ALT levels were classified as simple steatosis, whereas subjects with steatosis and elevated ALT levels (>40 IU/L) were classified as steatosis and abnormal ALT group.
A total of 748 children were enrolled, of whom 545 (73%) were normal weight, 110 (15%) overweight, and 93 (12%) obese. Two hundred and nineteen subjects then voluntarily received liver ultrasound examinations, including 134 from the normal-weight group, 44 from the overweight group, and 41 from the obese group (Fig. 1). After ultrasonographic examinations, children were divided into 2 groups, NAFLD and non-NAFLD, for analyzing the independent risk factors of pediatric NAFLD.
Statistical analysis was performed using SPSS version 13.0 (SPSS Inc, Chicago, IL). The χ2 test was used to compare categorical variables. One-way analysis of variance analysis with Bonferroni post hoc multiple comparisons were used to compare the differences among mean values of continuous variables among the subgroups. Multivariate logistic regression model was used to investigate the possible risk factors associated with NAFLD. The correlations between serum RBP4 levels and log value of variables were tested by simple and multiple linear regression analysis. A P value <0.05 was considered to be statistically significant.
Prevalence of NAFLD and Abnormal Liver Function in Schoolchildren
Forty-six (21%) of the 219 subjects were diagnosed as having NAFLD by ultrasound. The rate of NAFLD increased progressively from 3% in the normal-weight group to 25% in the overweight group, and 76% in the obese group (Fig. 1). Twenty-three (3%) of the 748 participants were found to have abnormal ALT values; 3 (0.6%) were in the normal-weight group and 20 (22%) were in the obese group. Of all the sera with an abnormal ALT value, none was positive for hepatitis B surface antigen or antihepatitis C virus antibody. We also observed that an abnormal ALT value was significantly more common in boys than in girls (30% vs 0%, P = 0.001) among children with obesity.
Anthropometric and Metabolic Characteristics of the Study Children
The baseline characteristics of the study children receiving ultrasound examinations are shown in Table 1, separated into non-NAFLD, simple steatosis, and steatosis with abnormal ALT. Among the 46 children with NAFLD, 14 (30%) had abnormal ALT values and were assigned to the steatosis and abnormal ALT group. The participants with simple steatosis had significantly higher age, BMI, TG, insulin, HOMA, ALT, and RBP4 levels than those of the normal controls (P < 0.05). In contrast with the normal controls, the children with steatosis and abnormal ALT had a significantly higher male ratio, BMI, TG, insulin, HOMA, AST, ALT, and RBP4 levels (P < 0.05). In comparisons of the anthropometric and metabolic characteristics between the subjects with simple steatosis and steatosis and abnormal ALT, there was a significantly younger age, and higher male ratio, BMI, insulin, HOMA, AST, and ALT levels in children with steatosis and abnormal ALT than those with simple steatosis (P < 0.05). In contrast, children with simple steatosis or steatosis and abnormal ALT had significantly lower HDL level than controls (P < 0.05); however, RBP4 levels were not different between the steatosis and abnormal ALT group and the simple steatosis group (27.8 vs 26.0 mg/L, P = 0.317).
Risk Factors of Pediatric NAFLD
The characteristics of the selected variables among the subjects with and without NAFLD are summarized in Table 2. BMI, TG, insulin, HOMA, AST, ALT, and RBP4 levels were significantly higher in the children with NAFLD as compared with those without (P < 0.001). The mean age of NAFLD children was also higher than that of non-NAFLD (P = 0.002); however, the level of HDL was significantly lower in NAFLD children than in non-NAFLD (P < 0.001). Total cholesterol and glucose levels were not statistically different between the 2 groups.
To assess the potential role of the obtained factors as predictors of NAFLD, multiple logistic regression analysis was computed (Table 3). We used NAFLD as the dependent variable, and excluded the highly correlated variables such as insulin to HOMA and AST to ALT. Finally, BMI (P < 0.001), serum RBP4 (P = 0.048), and ALT (P = 0.048) levels were independently significant predictors of fatty liver disease in children.
Correlation of RBP4 Levels with Clinical and Laboratory Variables
Serum RBP4 levels were identified to have a positive correlation with BMI (R2 = 0.052, P < 0.001), ALT (R2 = 0.052, P < 0.001), HOMA (R2 = 0.046, P = 0.001), insulin (R2 = 0.049, P = 0.001), and TG (R2 = 0.143, P < 0.001) but not HDL (R2 = 0.007, P = 0.28) levels in the simple linear regression model (Fig. 2). Glucose and AST levels were not correlated to RBP4 levels (P > 0.05). Moreover, multiple linear regression analysis confirmed that only serum TG levels were significantly related to RBP4 levels (P < 0.001).
The worldwide prevalence of childhood obesity has increased enormously during the last 3 decades (24,25). Since 2000, there have been studies reporting that approximately 15% of children and adolescents are typically overweight and a further 12% to 15% obese (25). We also identified that the prevalence of NAFLD among the obese participants was as high as 76%. These findings indicate that NAFLD may become the most common cause of pediatric chronic liver disease in Taiwanese children as in industrialized countries.
As in our study, several studies have reported that 14% to 24% of asymptomatic obese children were found to have abnormal ALT values (2,13,26,27). This feature was associated with insulin resistance and higher levels of TG (27,28). Therefore, hepatic steatosis is also considered to be a core feature of metabolic syndrome. To control the subsequent metabolic complications of obesity, researching the risk factors of NAFLD in children deserves attention. Previous studies have reported that the occurrence of nonalcoholic steatohepatitis in obese children is predominantly male (2,27). In the present study, 30% of the children with NAFLD showed an abnormality in ALT, and they were exclusively male. The cause for a sex-based difference in abnormal ALT values among children with NAFLD is unknown. One factor is that upper limit of normal ALT is higher in boys than in girls (29). Some reports have suggested that sex hormones influence the distribution of both fat and muscle. Another hypothesis is that sex hormone–binding globulin, produced in the liver, is potently correlated with insulin sensitivity (2,30).
Increased body adipose mass is a major risk factor for the development of human metabolic and cardiovascular disorders (15). Our results confirmed that BMI, TG, insulin, HOMA, AST, and ALT levels were significantly increased in children with NAFLD compared with normal controls (13,26). In addition, NAFLD children had a lower HDL than non-NAFLD (31). Advanced metabolic complications may occur after progressive liver injury, and we also demonstrated a stepwise increment of BMI, insulin, HOMA, and ALT levels from children with normal livers to simple steatosis, and to steatosis and abnormal ALT. This implies that health care providers should pay more attention to children with increment of BMI and ALT levels to prevent development of NAFLD and further metabolic complications.
In obese adults and children, serum RBP4 has been reported to be a contributor to insulin resistance (16,18,19). Recent studies have implied a role for RBP4 as a contributive or protective factor to NAFLD. Nevertheless, the available data on this issue are limited, particularly in children (20,21,32,33). As abnormal liver function was an adverse consequence of fatty liver disease, we found that children with NAFLD were independently associated with higher BMI and serum RBP4 levels as compared with non-NAFLD controls, but serum RBP4 levels were not significantly different between children with simple steatosis and steatosis with elevated ALT. Our results are different from the data presented by Nobili et al (20), who found that RBP4 levels were inversely correlated to the histological severity of nonalcoholic hepatic damage in children. The disagreement with our findings may be related to the selection of a population with differing severity (histopathology vs sonography and ALT) and duration of liver disease; however, they did not report comparisons between subjects with NAFLD and healthy controls by serum RBP4 levels. RPP4 may behave differently or be of different clinical import at different stages of liver involvement in the presence of obesity and insulin resistance. We also confirmed that serum RBP4 levels were significantly correlated to TG levels. This indicates that RBP4 level is linked not only to obesity and insulin resistance (16) but also to obesity and hypertriglycerides (18). It is slight discouraged in application of RBP-4 for clinical practice because there is no normal cutoff value in human. Therefore, early detection of increasing RBP4 level in children at risk predicts to control pediatric NAFLD and the metabolic disorders in future.
Because the presence of abnormal serum liver function tests does not well correlate with the pediatric histological liver damage (34), there is a strong need for studying the noninvasive predictors of NAFLD among children at risk. There are 2 major limitations to the present study. First, we used ultrasound examinations to identify fatty liver and elevated ALT to define the severe liver damage without histological confirmation. Liver histology is the criterion standard for assessing hepatic steatosis and steatohepatitis (8), but it is not appropriate or practical to perform liver biopsies on obviously healthy subjects. Alternatively, ultrasound scanning also provides sufficient specificity and sensitivity for the diagnosis of NAFLD (35). Second, the causal relation between NAFLD and the RBP4 level could not be determined because of the cross-sectional analysis.
In conclusion, the present study strongly indicates that elevated RBP4 and ALT levels as well as obesity are independent risk factors in developing pediatric NAFLD in Taiwan. It also suggests that serum RBP4 may be a valid marker of hypertriglyceridemia in children with NAFLD.
The authors thank Dr Solomon Chih-Cheng Chen (Department of Paediatrics, Chia-Yi Christian Hospital, Taiwan) for constructive help with the statistical analysis.
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child; hypertriglyceridemia; nonalcoholic fatty liver disease; obesity; retinol-binding protein 4
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