ZIMBA: Clinical Trial in Paediatric Obesity
ZIMBA: Clinical Trial in Paediatric Obesity
Myoinositol (MI) and D-chiro inositol (DCI) are isomeric forms of inositol that were found to
 have insulin-like properties, acting as second messengers in the insulin intracellular
 pathway; both of these molecules are involved in the increasing insulin sensitivity of
 different tissues to improve metabolic and ovulatory functions. Myoinositol is the
 predominant form that can be found in nature and food.

 Inositol has been mainly used as a supplement in treating several pathologies such as
 polycystic ovary syndrome (PCOS), metabolic syndrome, type 2 diabetes mellitus (T2DM) and
 gestational diabetes (GDM). In the case of GDM, a condition defined as a glucose impairment
 first detected in pregnancy, a preventive role of inositol for GDM onset was recognized. In
 addition, inositol has been studied as a therapeutic option for the treatment of GDM and
 T2DM. The main effect of inositol is decreasing the level of insulin resistance.
 Consequently, a potential role of inositol as a treatment option could be hypothesized for
 other conditions typically characterized by insulin resistance like metabolic syndrome and
 obesity.

 Zinc also plays an important role in insulin action and carbohydrate metabolism. It may also
 have a protective role in the prevention of atherogenesis. Several human studies have
 demonstrated that Zinc supplementation reduces total cholesterol, LDL cholesterol and
 triglycerides, in addition to increasing the HDL cholesterol levels. Studies have shown that
 diabetes is accompanied by hypozincemia and high levels of Zinc in urine. In addition Zinc is
 also an integral part of key anti-oxidant enzymes and Zinc deficiency impairs their
 synthesis, resulting in increased oxidative stress.

 A supplementation with Myo-Inositol and Zinc could represent a valid strategy in paediatric
 obesity in addiction to a standard approach. The purpose of our study is to evaluate the
 supplementation of Myo-inositol and Zinc in the treatment of 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:

 1. 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);

 2. 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);

 3. 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.