# Salivary Extracellular DNA and DNase Activity in Periodontitis
## 5 Discussion  
Saliva and its content can reflect health status. As the latest research shows, saliva contains various markers helpful for the diagnosis and monitoring of many disorders such as cancer [22; 23], neurological diseases [24], renal diseases [25], inflammatory bowel disease [26] or cardiovascular diseases [27]. The health status of the teeth, gingiva or salivary glands is also reflected in saliva [28]. Markers of inflammation, such as cytokines and inflammatory mediators, are produced as a result of bacterial infection and were proved to be higher in saliva of patients with periodontitis in comparison to healthy controls [29]. Based on the above-mentioned studies, total ecDNA or ecDNA of different origin were expected to be increased in periodontitis and could serve as a possible biomarker of periodontitis severity. However, as is defined by [PERSON] et al. [21], ecDNA is obtained after 16,000\(\times\)\(g\) centrifugation, although they used plasma, not saliva. In our previously published study [30], we tried to determine the total DNA in whole saliva as well as in salivary supernatant. We hypothesized that we have analyzed ecDNA once the fluid is centrifuged and depleted of cells. However, no differences were found either in ecDNA between healthy and chronic periodontitis, and similarly no differences were observed between genders. These results were possibly obtained due to technical issues such as insufficient centrifuging while analyzing ecDNA. Therefore, we decided to repeat this observational study, in which we obtained ecDNA appropriately prepared using two-step centrifugation, 1600\(\times\)\(g\) followed by 16,000\(\times\)\(g\) according to [PERSON] et al. [21]. It is assumed that, after 1600\(\times\)\(g\) centrifugation, only cells are spun down. However, generally, when talking about ecDNA, 16,000\(\times\)\(g\) centrifugation is needed in order to spin down the bigger particles and cell debris. Based on this, we decided to analyze DNA in all three fractions, i.e., in whole saliva, centrifuged saliva and double-centrifuged saliva. According to our results, total DNA was higher in patients with periodontitis in whole saliva perhaps due to increased cell debris as a result of bleeding and worsened oral health. Once the whole saliva was centrifuged, the differences in total DNA were not observed anymore. Also, when we quantified separately ncDNA and mtDNA, we could observe differences in mtDNA, but not in ncDNA. Increased concentration of mtDNA in periodontitis indicates the presence of mitochondria, which could potentiate the inflammation in oral cavity [31]. Mitochondria are more likely to be still intact, because after the double-centrifugation, the mtDNA concentrations are same in both groups. EcDNA of mitochondrial origin is the same in healthy controls as well as in periodontitis, however ecDNA of nuclear origin is increased in healthy controls, even though there are no differences observed in total ecDNA. This could happen due to higher fragmentation of ncDNA in periodontitis [30], since short fragments are not detectable by qPCR but are visible by spectrofluorometry. Moreover, fragmented DNA could further potentiate inflammatory status [21; 32; 33], and thus worsen the course of disease. This hypothesis should be clarified by further studies. Nevertheless, if this hypothesis is proven correct, the ecDNA might become another therapeutic target. We expected that altered DNase activity could be potentially associated with periodontitis. There was not significant change in DNase activity in our samples, however studying the activity of this enzyme is still in process as it is still not clear if the DNase activity is related with ecDNA concentrations. Even though the preliminary experiments on DNase activity have been already published [34], it is also not clear whether differences exist among genders under physiological circumstances [35]. However, it is important to remark that not all ecDNA is similarly sensitive to DNase activity.