Study Design
A case-control study design was adopted in this study. Obese and non-obese school children of the age group (6-11 years) were the subjects of interest in this study. The levels of salivary metabolic hormones – Ghrelin, Leptin, Adiponectin and Insulin in obese and non-obese subjects were assessed and compared to determine the significance of these hormones as biomarkers of childhood obesity.
Ethical Approval
All procedures performed in this study were in accordance with the ethical standards of the institutional and national research committee (Indian Council of Medical Research) and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Institutional Ethics Committee of Sri Ramachandra Institute of Higher Education and Research (IEC-NI/15/FEB/45/07). Written informed consent was obtained from the parents of the children who were willing to allow their children participate in the study.
Setting
The study was conducted in an elementary school and a summer campsite in Chennai, India, from July 2017 to March 2018, as part of an extensive research in the area of salivary appetite regulatory hormones and an intervention program targeting these hormones in obese children. Permission was obtained from the concerned authorities of the elementary school and the summer campsite for conducting the study. Anthropometric assessment of all children aged between six and eleven years was carried out at the sample sites to categorize them into obese and non-obese groups. Children who gave written informed parental consent and assent to participate in the study alone were recruited for the study as per the sample size recruitment.
Participants
A total of 1432 children from an elementary school (n=1378) and a summer campsite (n=54) were assessed for childhood obesity, using standard protocols. Of the 1432, 166 children (83 obese and 83 non-obese) were recruited for this study based on inclusion and exclusion criteria and consent to participate in the study. The criteria for inclusion were – i) children aged between six and eleven years categorized as normal (control) and obese (case) using the WHO growth reference for school aged children (2007) and, ii)children who gave written informed parental consent and assent to participate in the study. The criteria for exclusion were – i) children who had an obvious underlying medical cause of obesity (evaluated using a questionnaire) and, ii) children who had undergone any medical or nutritional therapy for obesity in the past six months.
Tools Used –
Anthropometry
Anthropometric measurements such as height and body weight of the subjects were assessed using standard protocols. A calibrated stadiometer was mounted on a wall and the height (in centimeter (cm)) of each subject was measured in an upright standing position on an even surface adjacent to the wall. Body weight of each subject was measured using a body fat analyzer (TANITA UM-076) and recorded in kilogram (kg). The readings were entered in the nutritional survey module of WHO AnthroPlus v.1.0.4 (2009) software. The height-for-age ‘Z’ (HAZ) scores, weight-for-age ‘Z’ (WAZ) scores, and body mass index-for-age ‘Z’ (BAZ) scores of the subjects were calculated using the WHO AnthroPlus software and the subjects were categorized into obese and non-obese categories based on their BAZ scores.
Salivary Analysis
Collection of samples of Saliva
On the day of sample collection, the subjects were asked to come to the sample site in a state of fasting. The subjects were made to rinse their mouth with water before sample collection. Saliva was collected using sublingual cotton roll technique (cotton roll placed under the tongue of the subject for a minute). Using a sterile forceps, the cotton roll was transferred to a 50 mL syringe and injected into a vacutainer tube, to collect approximately two milliliter of saliva from each subject. The samples collected in vacutainer tubes were arranged in a 96-vial storage rack placed in a freezer box. The principle investigator shifted the samples stored in the freezer box to the testing laboratory under safe conditions within 24 hours of sample collection. The samples were stored at -20°C and analyzed within a period of six months.
Multiplex Analysis of Salivary Markers
Magnetic Luminex® Assays were used to assess the concentration of the selected hormones in each sample. The assays (166 saliva samples) for three biomarkers leptin, adiponectin and insulin were performed on 50 µl of saliva sample using premixed 3-plex magnetic bead panels on a Bioplex 200 platform with no dilution. The assay procedure was carried out following the manufacturers’ protocol. The results were read within 90 minutes post assay and evaluated using an analyzer.
Ghrelin Assay
As inclusion of ghrelin in the premixed kit used for the other markers was not possible due to the interference in magnetic bead regions, a separate test kit (RayBio® Ghrelin Enzyme Immunoassay (EIA) Kit: EIA-GHR, EIAM-GHR, EIAR-GHR) was used to assess ghrelin in 100 µl of each saliva sample. The assay employed an in vitro quantitative technique for detecting ghrelin peptides based on competitive enzyme immunoassay principle. The assay procedure was carried out following the manufacturers’ protocol. A standard curve of known concentration of Ghrelin peptide in the samples was assessed.
Sample size
166 (Obese, Cases = 83; Non-obese, Controls = 83) was the calculated sample size, assuming μ1=48.0 and μ2 = 63.1; Difference of means = 24.6, based on the study conducted by Gil et al., 2009 [5], at a Power of 80%. The sample size was calculated using Piface by Russell V. Lenth. Version 1.76 – 29 June 2011.
Statistical analysis
All descriptive statistics are expressed using mean and standard deviation after analyzing the data for normality, confirming the normal distribution of data. Independent sample t-test was used to test the mean difference between the control and case groups. Box and whisker plots were drawn to display the variation in hormone levels of the subjects through the quartiles of data. Univariate binary logistic regression was used to calculate the risk factor analysis. Odds ratio was calculated to evaluate the odds of salivary metabolic hormones as biomarkers among the obese and non-obese groups and to imply an association between the salivary hormones and childhood obesity. The statistical analyses were done using SPSS version 23.0 and any p - value less than 0.05 was considered as statistically significant.