The oral cavity constitutes a unique environment in which the saliva and mucosa play a key role. The properties and components of saliva are important for maintaining oral health and protecting teeth. Saliva analysis is non-invasive and has a great potential for clinical use. It can be used to detect biomarkers of disease states in the oral cavity, but also in systemic diseases (1, 2). Carbonic anhydrases are a group of metalloenzymes, with a zinc atom as the active center, which are responsible for catalyzing the reversible reaction of carbon dioxide hydration (CO₂ + H₂O ? HCO₃^- + H⁺). According to Lindskog and Nyman (3), 16 different carbonic anhydrases occurring in mammals are currently known, differing in terms of enzymatic properties, place of expression and amino-acid sequence (4). Anhydrases are involved in the regulation of pH and the ionic and acid-base balance in various tissues of the body. The only secreted form is carbonic anhydrase VI (CA6), secreted mainly by the parotid and submandibular glands (5). CA6 is a salivary enzyme and may influence its properties such as pH regulation and buffering capacity, although its role is still not fully understood. A study by Kivela et al. (6) challenged the association of pH regulation and buffering capacity with high levels of CA6 in saliva. On the other hand, Kimoto et al. (7) demonstrated the ability of anhydrase to penetrate dental plaque and neutralize acids through bicarbonate buffers. Dušan et al. (8) suggests the usefulness of CA6 activity in saliva as a biomarker of caries disease. However, further research is required to confirm these reports.

The aim of the following study was to determine the relationship between the concentration of salivary carbonic anhydrase VI and various conditions in the oral cavity (tooth decay, hygiene status and gingivitis), physicochemical properties of saliva and the cariogenic bacteria count.

The study conformed to the STROBE guidelines (STrengthening the Reporting of OBservational studies in Epidemiology). The approval of the Bioetical Comission of Warsaw Medical University (No. KB/194/2015) was obtained.

Eighty-seven patients aged 12-16 years with permanent dentition took part in the investigation. The participants were recruited from Department of Pediatric Dentistry, Medical University of Warsaw. The inclusion criteria included no systemic diseases, no chronic medication intake, no developmental defects of tooth hard tissues. Patients representing those features were eliminated from the study.

Patients were examined in a dental office on the basis of standardized WHO criteria (9), by one researcher. The presence of dental caries was assessed as DMFT/DMFT index and the presence of white spot lesions (WSL) was evaluated. The assessment of the hygiene status was based on the ODI-S soft deposit index (component of the OHI-S index according to Greene and Vermillion) (10) and the approximate plaque index (API%) according to Lange (11). The gingival condition was determined on four surfaces of permanent teeth 16, 12, 24, 36, 32, 44 using the GI (Gingigal Index) according to Löe (12).

Saliva samples were collected from all participants in the morning (8.00-10.00), on an empty stomach, using commercial Salivette kits (Sarstedt, Nümbrecht, Germany). The swabs were inserted into the mouth and left there for 5 minutes, allowing the accumulating saliva to soak in. After collection, the samples were immediately placed in a refrigerator at + 4°C and centrifuged within 2 hours after collection. Centrifugation was performed according to the parameters indicated by the manufacturer (1000 x g, at a temperature of + 4°C, for 15 min). The unstimulated saliva flow was calculated by dividing the volume of saliva obtained from the patient after centrifugation by the sampling time. Then the samples were frozen at -80°C until further analysis.

The commercial GC Saliva Check Buffer (GC Europe) kit was used to assess the physicochemical properties of saliva (pH, buffer capacity, stimulated saliva flow, consistency), according to the manufacturer’s instructions. Salivary levels of cariogenic bacteria – Streptococcus mutans (SM) and Lactobacillus spp. (LB) were assessed with the commercial CRT Bacteria test (Ivoclar Vivadent), according to the instructions. Values were expressed as colony forming units per milliliter of saliva (CFU/ml).

Prior to testing, the samples were brought to room temperature. Freeze/thaw cycles were avoided. Laboratory testing was performed according to the protocol outlined in the ELISA kit for Carbonic Anhydrase VI (Cloud-Cone Corp.) and read at 450 nm on a BioTek Synergy™ Mx microplate reader (BioSPX B.V.).

The analysis was carried out with the use of the Statistica 12.0 program and the R package. Descriptive statistics characterizing their variability were calculated for quantitative variables. Proportions (%) with division into groups were calculated for binomial variables. The comparison of quantitative variables between the two groups was performed using the Mann-Whitney test and the chi-square test, while the comparison of three or more groups was performed using the ANOVA test. In the case of categorical variables (including binomial variables), the chi-square test was used to compare the groups. Spearman’s rank correlation coefficient was calculated. In all analyzes, the level of statistical significance was set at 0.05. Missing data was excluded from the analysis.