The study was approved in the research center of the Riyadh Elm University (FPGRP/43735004/277). Informed consent was obtained from the parents of children before saliva collection and verbal consents were obtained from the participating children before the collection of saliva was used in the study. Confidentiality of the participant was maintained throughout the study.
A sample power calculation was performed using the G-power sample power calculator ver. 3.1 ( Universtat Kiel, Kiel, Germany). It was estimated that for a power of 0.95, with an effect size of 0.8 a minimum of 20 children per group was needed to perform a One-Way ANOVA.
Children with BA were selected from the pediatric clinics of the Security Forces Hospital, Riyadh Saudi Arabia.). The inclusion criteria were that the participants were aged 6–8 years with bronchial asthma and (for group 1) childhood caries. Participants who were free of BA were selected from the dental clinics of Riyadh Elm University. The control and experimental groups were matched with each other according to age, gender, and extent of dental problems. A total of four groups were divided according to caries:
Group 1: The participants in this group had 4 or more dental caries lesions in the primary teeth (with or without dental caries on the first permanent molar); had at least one asthmatic attack in the past three months and were currently on Albuterol, Salbutamol or Terbutaline but were not on any other medication.
Group 2: The participants in this group were free of any dental caries and had at least one episode of asthmatic attack in the past three months. They were currently on Albuterol, Salbutamol or Terbutaline but were not on any other medication.
Group 3: The participants of this group had four or more active dental caries lesions in the primary teeth (with or without dental caries on the first permanent molar) and had no history of BA.
Group 4: The participants in this group were free of any dental caries and had no history of BA
Dental caries profile of the primary teeth was measured using the decayed filled teeth index (dft) and the dental caries profile of the permanent was measured using the Decayed, Missing Filled permanent teeth index (DMFT). Oral hygiene was evaluated using the oral hygiene index-simplified (OHI-S). The teeth were examined using WHO category II criteria – clinical examination with dental unit light and without radiographs. Data collection was conducted by one examiner and one data recorder (EK).
Saliva was allowed to pool in the floor of the mouth by asking the child to touch the chin to the chest for approximately one minute. Saliva was then collected using a sterile Pasteur pipette and transferred to a 1.5 ml Eppendorf tube. These sterile plastic collection tubes were closed carefully and placed in a 2-80C ice storage box to be transferred to the deep freezer of a ˉ600C until they could be analyzed. CA6 assays were performed using the zymographic method using a Human CA6 ELISA kit (Abbexa Ltd, Cambridge Science Park, and Cambridge, CB4 0EY, UK).(14)
The Shapiro Wilk test of CA6 levels showed a Kurtosis of 0.456 and skew of -0.01, suggesting that the sample was normally distributed. Therefore, parametric statistics were used. The difference in salivary carbonic anhydrase between the test and control groups was measured using the one-way analysis of variance (ANOVA) and Scheffe’s post hoc test A linear regression model using the CA-VI levels; a dependent variable and gender, caries and asthmatic medication as independent variables were designed. All Statistical analyses were performed using the statistical package for social sciences (SPSS) 25 data management software (IBM SPSS, Armonk, NY, USA).