## 4 Discussions  
In our study, the rapid test showed an increase in total nitrite in metal appliance wearers similar to ELISA for both T\({}_{1}\) and T\({}_{2}\) moments, as demonstrated by the statistical analysis. Moreover, the total nitrite concentration determined by the two methods (ELISA and Nitric Oxide Saliva Test Strips) displayed a strong correlation at all time points and for all three groups, suggesting that rapid testing could be used in daily practice as a substitute for a more expensive and laborious method with similar results. Oxidative stress determined by free radicals mainly affects the cellular lipid component and is the subject of current research, although results are conflicting. This study also found a difference between the inducible nitrate levels in the metal bracers sub-group, compared to the sub-group treated with thermoplastic aligners, suggesting a higher impact of the metallic bracers on the oral cavity oxidative status. Nitric oxide is a free radical that plays an important role as a regulator of various physiological and pathological mechanisms in the body. In the oral cavity, it may act as a non-specific antimicrobial defence system by inhibiting bacterial growth or by enhancing macrophage-mediated cytotoxicity. There are two main transformation mechanisms: reduction in the enzyme reductase, released by certain bacteria, which converts nitrate (NO\({}^{3-}\)) to nitrite (NO\({}^{2-}\)); or by facilitated reduction by certain bacterial products resulting from the microflora of dental plaque, forming nitric oxide and nitric acid (which ionises to NO\({}^{3-}\)). Enzymatically, it can also be produced from L-arginine by nitric oxide synthase (NOS), which has three isoforms: neuronal NOS (nNOS; Type I, NOS\(-\)1); inducible NOS (iNOS; Type II, NOS-2), which is present in various cells and tissues and is produced by immunocompetent cells--such as  
Figure 1: Correlations between total nitrite determined by ELISA and by the semi-quantitative method at T\({}_{0}\), T\({}_{1}\), T\({}_{2}\), T\({}_{3}\). macrophages infected by the bacteria involved in regulating inflammatory reactions; and endothelial NOS (eNOS; Type III, NOS-3), which is found in vascular endothelial cells [11]. iNOS levels are significantly increased following tissue trauma (when cells are activated by external stimuli) and probably participates in the development of periodontal disease [11]. Regarding NO levels, [PERSON] et al. showed similar mean values in patients with different oral cancers compared to orthodontic fixed appliances wearers [12]. Similarly, salivary NO levels in smokers are significantly higher than in non-smokers. In oral pathologies such as [PERSON]'s disease and recurrent aphthous stomatitis, the elevated salivary values may guide the clinician as to the degree of severity and reflect serum values [11; 13]. According to [PERSON] et al., nitric oxide levels do not show considerable changes during one year of wearing orthodontic fixed appliances. [PERSON] et al. demonstrated increases in NO levels from day 7 to day 30 since treatment initiation. In our study, total, endogenous, and inducible nitrite concentrations were determined in patients with fixed metal appliances and aligners and compared to a control group. The results revealed a statistically significant increase in total and inducible nitrite in the case of fixed metal appliances, compared to aligners and the control group. This elevation can be considered a physiological response to the presence of foreign bodies in the oral cavity [14; 15]. A potential mechanism for the rise in inducible nitrates in the metal brackets sub-group is the interplay between the metal ions released by the device, and endogenous and bacterial metabolites that can lead to the formation of free radicals [16]. Furthermore, metal brackets have a more rigid and retentive surface that comes into intimate contact with oral soft tissues such as the cheeks, which can lead to the interaction of metal ions with surrounding cells, which, in turn, can lead to inflammation.