ZIMBA: Clinical 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, with pubertal stage ≥ 3 according to the Tanner stage, obese according to the IOTF criteria (International Obesity Task Force), diet naïve or with failure of weight loss (defined as -1 kg/m2 BMI in 1 year).

Intervention: Patients will be randomized in a open-label, into two groups homogeneous for number and sex of the subjects. One group (group "active") will receive the supplementation with Myoinositol and Zinc (active product) and the other group (group "Placebo") will receive a placebo for a total of 3 months of treatment.

Dietary restriction: The standard diet will be distributed with 55-60% of carbohydrates, 25-30% lipids and 15% proteins, and will be performed in accordance with the calories of an isocaloric balanced diet calculated throughout the Italian LARN Guidelines for age and gender (Italian Society of Human Nutrition, 2014), inspired to Mediterranean pyramid.

Physical activity: all subjects will receive general recommendations about performing physical activity.

Randomization: Participants will be randomly assigned in a 1:1 to active intervention Group (Active Group) or Placebo Group.

Timing: Patients will be evaluated firstly at time of enrollment (V0) and at the end of the end of the study (V1).

The following anthropometric measures, biochemical and ultrasound evaluations and questionnaires will be obtained:

• Anthropometric measures:

• height (V0, V1);
• weight (V0, V1);
• body mass index (BMI; Kg/m2) (V0, V1);
• waist and hip circumferences (V0, V1); for the calculation of the following ratios: waist/hip, waist/height;
• Tanner stage (V0, V1); (Tanner JM, 1961);
• blood pressure and heart rate (V0, V1);
• Biochemical evaluations (after a 12-h overnight fast): CBC (Complete Blood Count) with formula, serum insulin-like growth factor 1 (IGF1, ng/mL), 25-hydroxy (OH) vitamin D (ng/mL), uric acid (mg/dL), Serum Zinc (mg/dl), alkaline phosphatase (U/L), ACTH (adrenocorticotropic hormone) (pg/mL), cortisol (microg/dL), TSH (thyroid-stimulating hormone)(uuI/mL), fT4 (serum free T4) (ng/dL) (V0, V1); 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) (V0, V1); 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 (Dialysis Outcome Quality Initiative), the eGFR will be calculated using updated Schwartz's formula: eGFR (mL/min/1.73 m2) = [0.413 x patient's height (cm)] / serum creatinine (mg/dL)(V0, V1); 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)(V0, V1); 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(V0, V1); oral glucose tolerance test (OGTT: 1.75 g of glucose solution per kg, maximum 75 g) and samples will be 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 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) (V0, V1); 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) (albumin-creatinine ratio), will be calculated using the following formula: [urine albumin (mg/dL) / urine creatinine (mg/dL)] x 1000. A sample of serum and a sample of plasma will be collected at each time and will be stocked in -20°C freezer for further laboratory analysis (V0, V1);
• Nutritional and physical activity measurements:

• KIDMED questionnaire for children and adolescents (Serra-Majem L et al., 2004). 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, V1);
• 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 (V0, V1);

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 23 individuals has been estimated to be sufficient to demonstrate a difference of 2 points of HOMA-IR (Prodam F et al, 2013) with 90% power and a significance level of 95% and a drop-out rate of 10% 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.

Inclusion criteria

• both sexes
• between 6 and 18 years of age
• obese, according to the IOTF criteria (Cole TJ et al., 2000)
• pubertal stage ≥ 3 according to the Tanner stage (Tanner et al., 1961)
• HOMA-IR > 2,5 or insulin > 15 µU/ml
• Serum Zinc level in the range of normality or under the normal levels.

Exclusion criteria

• Adverse reactions to the product or component of the product (allergies…)
• Genetic obesity (Prader Willi syndrome, Down syndrome), Metabolic obesity (Laurence-Biedl syndrome…), endocrinological obesity (Cushing syndrome, hypothyroidism)
• Chronic diseases, hepatic or gastroenterological diseases
• Medical treatment for chronic diseases
• Supplementation with inositol-like products or supplements containing Zinc and Inositol.