Participants and setting
Data of 190 obese children and adolescents, aged 6 to 16 years old, were collected during the second semester of 2010. The participants were invited, through media advertisement, to participate in a community-based program. This program aimed to treat obesity focused on enhancing exercise and nutritional orientation and was carried out at facilities of the Universidade Estadual Paulista - Campus of Presidente Prudente (FCT/UNESP), São Paulo, Brazil.
The inclusion criteria were: (a) to be classified as obese according to the reference values proposed by Cole et al.[10], (b) aged between 6 and 16 years at the first evaluation date, and (c) participants and parents/guardians signed a written informed consent form for the participation in the program.
The Ethical Committee of FCT/UNESP approved this study (Protocol number 07/2009).
Anthropometry
Body weight (BW) was measured with an electronic scale (precision 0.1 kg [Filizzola PL 150, Filizzola Ltda]) and the height (H) with a wall-mounted stadiometer (precision 0.1 cm [Sanny®, São Paulo, Brazil]). Both measurements were carried out with participants wearing light clothing and no shoes. Body mass index (BMI) was then calculated as kg/m2. All anthropometric measurements were performed by trained researchers, according to standardized techniques[11].
Dual Energy X-Ray Absorptiometry
Body composition was estimated by a Dual-energy X-ray absorptiometry (DEXA) scanner (Lunar DPX-NT; General Electric Healthcare, Little Chalfont, Buckinghamshire), with software version 4.7. The method estimated the body composition by fractionating the body into three anatomical compartments: fat-free mass (FFM), fat mass (FM) and bone mineral content. The assessment was carried out in approximately 15 minutes, and the subjects remained still and in a supine position throughout the scan, wearing light clothing while lying flat on their back with arms by the side. The results were expressed in kilograms of FFM and FM. The absolute (kg) and relative (%) values from trunk fat (%TF) were also assessed by DEXA. All DEXA measurements were carried-out at the University laboratory in a controlled temperature room. Each morning, before the beginning of the measurements, the DEXA was calibrated by the same researcher, according to the references provided by the manufacturer.
Stages of puberty
The stage of puberty was self-assessed by the participants aged older than 10 years. A standardized series of drawings was given to the subjects to assess their own pubertal development. Girls received drawings of the five stages of Tanner breast and female pubic hair development with appropriate descriptions accompanying the drawings. Boys were given line drawings of boys showing the five Tanner stages of pubic hair development, with appropriate written descriptions. Participants were asked to select the drawing of the stage that best indicated his other own development. All procedures were done as described by Marshall & Tanner[12]. The results were placed by each subject in a locked box with only their subject id as an identifier, so as to guarantee the integrity and anonymity of the subjects. Only the main researcher had access to those questionnaires.
The breast stages for girls and genitalia stages for boys were chosen to classify the sexual maturation status because they are recommended by a World Health Organization Expert Committee as indicators of sexual maturation for international use[13]. The original five pubertal stages were grouped in three new groups: participants aged under 10 years and those who answered stage 1, were grouped in group 1; those who answered stages 2 and 3, were grouped in group 2; and those who answered stages 4 and 5, were grouped in group 3.
Ultrasound
The ultrasound equipment (Toshiba Aplio Model Tochigi-ken, Japan) was used by a trained radiologist to assess the level of fat accumulation and the morphology of the liver. Finally the NAFLD diagnosis was made by a hepatologist. The NAFLD was graded semi qualitatively as described by Shannon[14]: Score 0 is absence of steatosis, defined as normal liver echo texture; Score 1 is mild steatosis, defined as slight and diffuse increase in fine parenchymal echoes, normal visualization of diaphragm and portal vein borders; Score 2 is moderate steatosis, defined as a diffuse increase in fine echoes with slightly impaired visualization of diaphragm and portal vein borders and; Score 3 is severe steatosis, defined as fine echoes with poor or no visualization of diaphragm, portal vein borders and posterior portion of the right lobe. The participants were labeled as “without NAFLD” (N-NAFLD) and those with any of the above mentioned grade of fatty infiltration in the liver were labeled as “with NAFLD” (P-NAFLD).
The ultrasound is an image evaluation that provides diagnosis of NAFLD and measures, in centimeters, the length of intra-abdominal fat (IAF) and subcutaneous fat (SCF).
Blood samples
A 12-hour fasting blood sample collection was taken and analyzed by a private laboratory located in the city of Presidente Prudente, Brazil. Samples were collected in vacuum tubes containing gel with anticoagulant. Then the blood was centrifuged for 10 minutes at 3,000 rpm. To measure total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C) and glucose (GL), an enzymatic colorimetric kit processed in a unit Autohumalyzer A5 was used[15]. Insulin was analyzed using the RayBio® Human Insulin ELISA Kit, following the Manual Revised Nov 23, 2009.
Blood pressure
An automated digital blood pressure monitor (Omron Healthcare, Inc., Intellisense, modelo HEM 742 INT, Bannockburn, Illinois, USA) was used for the measurement of systolic (SBP) and diastolic (DBP) blood pressure. This equipment was previously validated for pediatric population[16]. After fifteen minutes of resting in a lying-down position, two measures were taken on the right arm, with a two minute interval between them. The mean value was used for statistical analysis.
Diagnosis of metabolic syndrome
For diagnosis of Metabolic Syndrome (MS), we adopted the cut-point proposed by the World Health Organization (WHO)[17] for adolescents. The MS was diagnosed in subjects who presented three or more of the following risk factors: Obesity (BMI > percentile 95 according Must[17]); Glucose Homeostasis (prepubertal hyperinsulinemia >15 mU/L;[18] [stage 1 Tanner] pubertal>30 mU/L[19]; [stages 2–4 Tanner] pospubertal ≥20 mU/L [stage 5 Tanner]; Fasting Glucose ≥6.1mM/L and Impaired Glucose Tolerance: 120min ≥ 7.8mM/L), Elevated Blood Pressure (SBP > perc95 for age, sex and height proposed by NHBPEP[19]). Dyslipidemia (Triglycerides >105mg/dL for children< 10 years and >136mg/dL for children ≥ 10 years, HDL-C <35 mg/dL and Total Cholesterol > percentile 95).
Data analysis
Descriptive data are shown as means and standard deviation. One-way analysis of variance (ANOVA) was applied to analyze the differences in body composition, blood pressure and biochemical variables between males and females within NAFLD groups. A multivariate analysis was used to compare all variables within NAFLD group as dependent variable. Data were adjusted for sex, age, pubertal stage and total fat mass. The relative risk (odds ratio) and 95% confidence interval were calculated by binary logistic regression. The statistical significance was set at 5% for all the analyses and the calculations were conducted using SPSS, version 17.0 (SPSS Inc. Chicago. IL).
