GOOD-DAY Trial in Paediatric Obesity

Study design: A single-center pilot open-label randomized control trial. Population: The study will comprise a total of 60 subjects of both sexes, between 10 and 18 years of age, obese, according to the IOTF criteria (Cole TJ et al., 2000) and with visceral adiposity, as waist circumference ≥ 90th percentile (Cook S et al., 2003 - Cruz ML, Goran MI, 2004 - de Ferranti SD et al., 2004), diet naïve or with failure of weight loss (defined as -1 kg/m2 BMI in 1 year).

Inclusion/ Exclusion criteria (see Eligibility Criteria). Intervention: Patients will be randomized in a open-label, into two groups homogeneous for number and sex of the subjects. One group (group SD) will receive Standard Diet according to routine care and practice, and one group (group SD+MSD) will receive a standard diet according to routine clinical practice + a 30 minutes Mediterranean-Style Diet educational training with explanation of Mediterranean pyramid + gamification to Mediterranean diet inside the Hospital and at home throughout "The Mediterranean Goose".

Dietary restriction: Thestandard diet will be distributed with 55-60% of carbohydrates (45-50% complex and no more than 10% refined and processed sugars), 25-30% lipids and 15% proteins, and will be performedin accordance with the calories of an isocaloric balanced diet calculated throughout the Italian LARN Guidelines for age and gender (Società Italiana di Nutrizione Umana, 2014), inspired to Mediterranean pyramid.The standard diet will be given as diet plans in writing, after explanation by a well-trained and experienced clinical paediatric endocrinologist.

Education in Hospital: As well as receiving the aforementioned recommendations, the SD+MSD group will also receive a simple visual guide of Mediterranean food pyramid explained by a dietitian/nutritionist for 30 minutes (Bach-Faig A et al., 2011).

Gamification: The MSD Group will be involved inside the Hospital in an educational and playful training playing to "The Mediterranean Goose" in the ludic area of the Pediatric Department; this game has been proposed in an educational project known as "Mediterranean diet and enhancement of traditional foodstuff - MedDiet", founded by European Union in the framework of the ENPI CBC Mediterranean Sea Basin Programme (www.med-diet.eu). This material will be given to the family for continue to play also at home.

Physical activity: all subjects will receive general recommendations about performing physical activity. Exercise will be conducted daily and will consist of 30 minutes of aerobic physical activity.

Randomization: Alternately per week, participants will be randomly assigned in a 1:1 to one of the two dietary intervention groups.

Timing: Patients will be evaluated firstly at time of enrollment (V0) and, during the first 2 weeks of study (V1), biochemical and ultrasound evaluations will be completed. During the year of intervention, patients will be evaluated after 3 months, 6 months and 12 months (V2, V3, V4).The following anthropometric measures, biochemical and ultrasound evaluations and questionnaires will be obtained:

1. Anthropometric measures:

• height (V0, V2, V3, V4);
• weight (V0, V2, V3, V4);
• body mass index (BMI; Kg/m2) (V0, V2, V3, V4);
• waist and hip circumferences (V0, V2, V3, V4) for the calculation of the following ratios: waist/hip, waist/height;
• Tanner stage (V0, V2, V3, V4) (Tanner JM, 1961);
• blood pressure and heart rate (V0, V2, V3, V4).

1. Biochemical evaluations (after a 12-h overnight fast): CBC with formula, serum insulin-like growth factor 1 (IGF1, ng/mL), 25-hydroxy (OH) vitamin D (ng/mL), uric acid (mg/dL), alkaline phosphatase (U/L), ACTH (pg/mL), cortisol (microg/dL), TSH (uuI/mL), fT4 (ng/dL) (V1, V3, V4); aspartate aminotransferase (AST, IU/L), alanine aminotransferase (ALT, IU/L); AST-to-ALT ratio will be calculated as the ratio of AST (IU/L) and ALT(IU/L)(V1, V3, V4); serum creatinine concentration (mg/dL) will be measured with the enzymatic method; according to the NKF-K/DOQI Guidelines for CKD in children and adolescents (Hogg RJ et al., 2003), the eGFR will be calculated using updated Schwartz's formula (Schwartz GJ et al., 2009): eGFR (mL/min/1.73 m2) = [0.413 x patient's height (cm)] / serum creatinine (mg/dL)(V1, V3, V4); glucose (mg/dL), insulin (μUI/mL); insulin-resistance (IR) will be calculated using the formula of Homeostasis Model Assessment (HOMA)-IR: (insulin [mU/L] x glucose [mmol/lL) / 22.5)(V1, V3, V4); lipid profile: total cholesterol (mg/dL), High-Density Lipoprotein (HDL)-cholesterol (mg/dL), triglycerides (mg/dL); Low-Density Lipoprotein (LDL)-cholesterol will be calculated by the Friedwald formula and non-HDL (nHDL)-cholesterol will be also calculated(V1, V3, V4); oral glucose tolerance test (OGTT: 1.75 g of glucose solution per kg, maximum 75 g) and samples willbe collected for the determination of glucose and insulin every 30 min. The area under the curve (AUC) for parameters after OGTT will be calculated according to the trapezoidal rule. Insulin sensitivity at fasting and during OGTT will be calculated as the formula of the Quantitative Insulin-Sensitivity Check Index (QUICKI) and Matsuda index (ISI). The stimulus for insulin secretion in the increment in plasma glucose as insulinogenic index will be calculated as the ratio of the changes in insulin and glucose concentration from 0 to 30 min (InsI). Βeta-cell compensatory capacity will be evaluated by the disposition index defined as the product of the ISI and InsI (DI) (Kahn SE et al., 1993)(V1, V4); a collection at rest of first-morning urine sample. Physical and chemical urinalysis; urine albumin (mg/L) will be determined by an advanced immunoturbidimetric assay and urine creatinine (mg/dL) will be measured using the enzymatic method. Urine albumin to creatinine ratio (u-ACR - mg/g), will be calculated using the following formula: [urine albumin (mg/dL) / urine creatinine (mg/dL)] x 1000. For these calculations both albumin and creatinine will be in the same unit. The subjects whose urine will be found positive, they will undergo a collection of two more samples and will be considered the u-ACR mean value of these (V1, V3, V4).
2. Ultrasound evaluations:

• liver ultrasound(V1, V4);
• echocardiography and carotid sonography(V1, V4).

1. Nutritional and physical activity measurements:

• KIDMED questionnaire for children and adolescents; the index comprises 16 yes or no questions (Serra-Majem L et al., 2004), and the total score of the KIDMED index ranged from -4 to 12 and is classified into 3 levels: 1)≥8: optimal Mediterranean diet; 2) 4-7: improvement is needed to adjust intake to the Mediterranean diet; 3) ≤3: very low diet quality; the Italian version is reported and approved by Istituto Superiore Sanità in Rapporti ISTISAN 12/42(Istituto Superiore della Sanità, Rapporti ISTISAN 12/42, 2012)(V0, V2, V3, V4);
• the Food Frequency Questionnaire section of the Children's Eating Habits Questionnaire (CEHQ-FFQ), performed by Identification and prevention of Dietary and lifestyle-induced health Effects In Children and infantS (IDEFICS) study, on which parents or other caregivers will be asked to report the number of meals the children usually consumed at home or at other people's homes, such as of grandparents and friends, in a typical week of the previous month (Huybrechts I et al., 2011) (V0, V2, V3, V4);
• 24-hour recall and a list of all foods and constituents included in the Dietary Inflammatory Index (DII) along with the respective adjusted score will be evaluated. Food- and constituent-specific scores will be multiplied by the intake for each subject. and then will be summed to create the overall Inflammatory Index score (Cavicchia PP et al., 2009 - Shivappa N et al., 2014) (V0, V2, V3, V4);
• the Mediterranean Adequacy Index (MAI) will be obtained by dividing the sum of the percentage of total energy from typical Mediterranean food groups by the sum of the percentage of total energy from non-typical Mediterranean food groups, according to the following equation (Alberti-Fidanza A, Fidanza F, 2004) (V0, V2, V3, V4);
• the International Physical Activity Questionnaire (IPAQ) developed, validated for Italian adolescents (IPAQ-A) will be developed; It covers four domains of physical activity: (1) school-related physical activity, including activity during physical education classes and breaks, (2) transportation, (3) housework and (4) leisure time (Hagstromer M et al., 2008 - Mannocci A et al, Accepted to Minerva Pediatrica) (V0, V2, V3, V4).

Outcomes (see Outcome Measures). Information retrieval: A case report form (CRF) will be completed for each subject included in the study. The source documents will be the hospital's or the physician's chart.

Statistical e sample size: A sample of 60 individuals (30 in each group) has been estimated to be sufficient to demonstrate a difference of 0.4kg/m2 in BMISDS with a SD of 0.65 (Prodam F et al, 2013) with 90% power and a significance level of 95% and a drop-out rate of 20% using the Student test. Statistical significance will be assumed at P< 0.05. The statistical analysis will be performed with SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL, USA).

Organization characteristics: The study will be conducted at the Pediatric Endocrine Service of Division of Pediatrics, Department of Health Sciences, University of Piemonte Orientale, in Novara.

All blood samples will be measured evaluated using standardized methods in the Hospital's Chemistry Laboratory, previously described (Prodam F et al., 2014 - Prodam F et al., 2016).

Good Clinical Practice: The protocol will be conducted in accordance with the declaration of Helsinki. Informed consent will be obtained from all parents prior to the evaluations after careful explanations to each patient.