Participants
A double-blind RCT was conducted during 2021–2022, involving adolescents with overweight or obesity referred to the Obesity Clinic of the Mofid Children’s Hospital, Tehran, Iran, who were selected based on inclusion and exclusion criteria. The ethics committee of the Iran University of Medical Sciences approved the study. Moreover, this clinical trial was registered on the Iranian Registry of Clinical Trials (www.irct.ir) website (IRCT20091114002709N57; registration date: 2021-06-20). The flow chart of the study design and the schedule of the project are shown in Fig. 1.
Inclusion and exclusion criteria
Inclusion criteria included the following items: 1) Willingness to cooperate and sign the informed consent form after full knowledge of the objectives and method of the study; 2) Adolescent girls and boys with overweight and obesity, aged 10 to 19 years; 3) body mass index (BMI) Z-score higher than 1 and less than 3 based on age and sex (according to the definition of World Health Organization (WHO) [24, 25]). Also, adolescents were not included in the study if they meet the following criteria: 1) Use of probiotic supplements, prebiotics, symbiotics or any foods fortified with these supplements during the last 3 months; 2) Use of any antibiotics for 3 months before the study; 3) History of type 1 or 2 diabetes, cardiovascular, hepatic, gastrointestinal (celiac disease, irritable bowel syndrome, and inflammatory bowel disease) or renal diseases and metabolic disorders including maple syrup urine disease and phenylketonuria, urea cycle disorders; 4) History of gastrointestinal surgery; 5) Use medications or supplements that affect appetite, weight, or metabolism at least 3 months before the study (such as medications that affect carbohydrate, protein, or fat metabolism, and also medications that reduce or increase appetite or food intake, including herbal supplements); 6) Adherence to weight loss diet or any type of heavy exercise program during the last 6 months; 7) Pregnant and lactating adolescents; 8) Smoking (more than one cigarette in the last week or more than 200 cigarettes in Lifespan); 9) Having any allergy to chitosan or crabs and shrimp. We also excluded those who with any acute illness, the occurrence of any accident that affects a person’s health, the use of antibiotics during the study, failure to follow the supplement based on personal reasons or other reasons, and migration. Furthermore, the admission rate of patients after the intervention period was calculated using the following formula, and patients whose admission rate is less than 80% were excluded from the study. Acceptance rate = number of packages received at the beginning of the study/number of packages consumed at the end of the study * 100.
Sample size calculation
Given the absence of a study that investigated the effect of chitosan on weight loss in children and adolescents with overweight or obesity, we used a method of Reinehr et al. in order to calculate the sample size by considering the BMI Z-score as the primary outcome, as these authors examined the effects of prebiotics on overweight and overweight children. In this way, considering the difference of 0.066 units in the mean BMI Z-score at the end of 12 weeks of intervention, assuming S1 = 0.07 and S2 = 0.09, and with the type I of error probability level of 5% (α = 0.05) and the type II error probability level of is 20% (β = 0.20, power = 80%), the number of samples was calculated based on the sample size formula below 24 participants in each group. Assuming 30% of the possible loss, 32 participants in each group and a total of 64 participants were included in our study.
Study design and intervention
In this randomized double-blind randomized clinical trial with 12 weeks of intervention, 64 adolescents with overweight or obesity who meet the inclusion criteria were randomly divided into two groups receiving chitosan supplement and placebo (maltodextrin). The appropriate amount of chitosan supplementation in most studies is approximately 3 g/d [26,27,28]. Since no toxicity of this substance has been reported to mammals in the FDA [29], the participants received 1.5 g (Twice a day a total of 3 g) of chitosan powder (intervention group) or maltodextrin (placebo group) daily 30 minutes to 1 hour before lunch and dinner for 12 weeks. Standard fruit flavorings were added to these supplements, taking into account the possibility of individuals not consuming raw chitosan powder. Parents were advised to add the recommended amount of powder for each person to 250 ml of water. The supplements were provided by Karen Pharmaceuticals and Vital-Food Supplements Company. The powders were given to the parents at the beginning of the study and at the end of the fourth eighth week, and they are asked to bring empty packages of cans at the end of the fourth, eighth and twelfth weeks to check the acceptance rate of supplements.
At baseline, all study participants received recommendations for gradual weight loss (0.5 to 1 kg per month). According to age, sex, height, and BMI Z-score, the energy consumption was calculated according to the formulas proposed in Krauss’s book and a slight reduction of 200 kcal per person is considered [30]. The caloric distribution of the diet was estimated at 30% fat (7% saturation), 50% carbohydrate, and 20% as protein and the maximum amount of cholesterol and 300 mg per day. Dietary recommendations were the same for both groups, and both groups are asked not to consume fortified sources of probiotics, symbiotics, or prebiotics or supplements during this study.
Randomization and allocation
To ensure the uniform distribution of the main variables can have a great impact on the results (BMI Z-score and gender) in two groups, we used random allocation by Stratified Randomization and Permuted block randomization method. Based on the sample size of present study (64 subjects), we produced the double block and quadruple block using the online site (www.sealedenvelope.com). At the beginning of the study, sets of packages containing chitosan powder were prepared by someone other than the researcher due to the double-blindness of the study, and the placebo was similar in appearance to chitosan powder. All of the researchers from the allocation of participants in each of the groups (intervention and control group) until the end of the intervention, were not know the groups whereby the patients were randomized. In order to apply concealment in the randomization process, we used unique codes which generated by the company receiving the supplements and placebos on the medicine boxes. So, none of the participants and researchers know which of the two groups received the supplement or placebo with this method. Upon each person entering the study, based on the sequence generated, the medicine boxes in which the code is recorded was assigned to the participants.
Evaluation of personal information
At the beginning of the study, personal information including name, age, sex, dietary supplements, and history other diseases were completed using the face-to-face interview technique (person or parents). Maturity status is determined by a trained individual using Marshall and Tanner tables [31].
Anthropometric and physical activity measurements
Anthropometric variables were measured before and at the end of the study. Adolescents’ height and weight were measured with minimal clothing and without shoes. The weight of all subjects was measured twice with the Seca digital scale (made in Germany) with an accuracy of 0.01 kg. The height of the participants in the study was recorded standing using a tape measure, without shoes and with an accuracy of 0.5 cm at the beginning and end of the study; measures were collected twice each time and their average was recorded). BMI was determined as weight in kilograms divided by height in meters squared. Patients’ waist circumference was measured using a non-elastic tape measure with a maximum error of 0.5 cm, considering the middle half of the body below the ribs of the chest.
The BMI Z-score, also known as the standard deviation for BMI score, is a measure of relative weight and height that is set for age and gender in a reference standard. These scores are considered more suitable for determining longitudinal changes in body weight and obesity, and are also a superior criterion for comparing the mean values of the group [32]. Therefore, BMI Z-score was used to assess changes in body weight in participants. The level of physical activity at the beginning and end of the study was assessed by the International Physical Activity Questionnaire (IPAQ) in Persian. The amount of physical activity was calculated as small continuous data taking into account the coefficients related to the activity, and recorded as Met-min/week. The Met coefficient for walking is 3.3, for moderate activity is 4, for heavy activity is 8, which is multiplied by the duration of the activity in minutes and the number of days in the activity week, and its sum as the amount of physical activity in the week is set [33].
Biochemical measurements
At the beginning of the study and at the end of the twelfth week, after 12 to 14 hours of fasting, 5 cc of a venous blood sample was taken from the patients while sitting on a chair. These samples were centrifuged at room temperature for 10 minutes at 3700 rpm to separate their serum. The isolated serum was placed in 1.5 cc microtubules to measure biochemical factors was stored in a freezer at − 80 °C until testing.
Serum total cholesterol and triglyceride (TG) levels were measured using Pars Azmoon commercial kits (Tehran, Iran) by a biochemistry autoanalyzer, and serum high-density lipoprotein cholesterol (HDL-C) were measured after deposition of apolipoprotein B-containing lipoproteins with phosphotungstic acid solution. In cases where the triglyceride level was less than 400 mg/ml, serum LDL-C levels were calculated using the Friedewald Equation [34]: LDL-C = total cholesterol -TG/6 - (HDL-C). In other cases, commercial kits were used as a surrogate measure.
Pars Azmoon commercial kits (Tehran, Iran) were used to measure fasting blood glucose (FBG) by a biochemistry autoanalyzer and serum insulin levels by the immuno-turbidimetry method. Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as fasting insulin (mU/L) × FBG (mmol/L)∕405. Serum NPY was assessed using an ELISA kit (Crystal Day Biotech Co, Shanghai, China). Leptin and adiponectin were measured by using an ELISA kit (Mediagnost Co, Germany).
Dietary assessment
Evaluation of dietary intake at the beginning and end of the study and each time using a 24-hour dietary recall questionnaire of 3 days (2 normal days and 1 day off) was done interviewing the adolescents or parents. Related data, including energy intake, macronutrients, and some micronutrients were determined by Nutritionist 4 software.
Statistical analysis
SPSS-24 software (IBM Corp. IBM SPSS Statistics for Windows, Armonk, NY) was used to obtain statistical analyses. Quantitative variables were reported as mean (standard deviation) and qualitative variables were reported as numbers (percentage). Because the data was not normal, Mann-Whitney test were used to compare the results between baseline and end of the intervention between groups. As well as Wilcoxon test, were used to analyze within-group data. ANCOVA test was used to estimate any differences in treatment group at the end of trial with adjusting for covariates. Also, Chi-square test were used to compare qualitative factors. Significant levels for all tests were considered as P-value < 0.05.