i) 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.
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.
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.
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
Children who gave written informed parental consent and assent to participate in the study.
iv) Tools Used
Anthropometric measurements such as height (cm) and body weight (Kg) of the subjects were assessed. 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 WHO AnthroPlus v.1.0.4 (2009) and the subjects were categorized into obese and non-obese categories based on their BAZ scores.
b) Salivary Analysis
Collection of samples of Saliva
On the day of sample collection, the subjects (in fasting state) 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. Saliva samples were thawed every time before assay procedures. The kit components were brought to room temperature and reagents were prepared as instructed (wash buffers, beads, standards, etc.). Assay plates (96-well) with assay buffer, standards, samples and beads were covered and incubated on a horizontal orbital microplate shaker (800±50 rpm) for two hours at room temperature. Plates were washed and detection antibody cocktail was added. The plates were covered and incubated for one hour at room temperature on the shaker set at 800 ± 50 rpm. Streptavidin-phycoerythrin fluorescent reporter was added to all wells and the covered plate was incubated for 30 minutes at room temperature on the shaker set at 800±50 rpm. Plates were washed thrice and beads were resuspended in wash buffer and incubated on the shaker for two minutes at 800 ± 50 rpm. The results were read within 90 minutes and evaluated using an analyzer.
As inclusion of ghrelin in the premix used for the other markers was not possible, 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. In this assay, a biotinylated ghrelin peptide was spiked into the samples and standards. The samples and standards were then added to the plate, where the biotinylated ghrelin peptide competed with endogenous (unlabeled) ghrelin for binding to the anti-ghrelin antibody. After a wash step, any bound biotinylated ghrelin then interacted with horseradish peroxidase (HRP)-streptavidin, which catalyzed a color development reaction. The intensity of the colorimetric signal was directly proportional to the amount of captured biotinylated Ghrelin peptide and inversely proportional to the amount of endogenous Ghrelin in the standard and samples. A standard curve of kown concentration of Ghrelin peptide was established and the concentration of Ghrelin peptide in the samples was assessed.
v) 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  and a Power of 80%. The sample size was calculated using Piface by Russell V. Lenth. Version 1.76 – 29 June 2011.
vi) Statistical analysis
All descriptive statistics are expressed using mean and standard deviation. Independent sample t-test was used to test the mean difference between the control and case groups. 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.