**4. Discussion**

This study evaluated the association of different conditions such as gingival health, periodontitis, CAD, or a combination of both diseases (periodontitis and CAD) on saliva and serum vitamin C levels. This study found that periodontitis in CAD patients was associated with decreased levels of serum and salivary vitamin C and hs-CRP levels. However, compared to periodontitis and healthy subjects, only patients with CAD and periodontitis + CAD presented significantly lower salivary and serum vitamin C levels, supporting the hypothesis that CAD may have contributed to decreased serum and salivary vitamin C levels.

Moreover, our results showed that the presence of periodontitis in patients with CAD might serve as an inhibitor of vitamin C and for associated risk of CAD and CVD. Recent investigations suggested that low serum vitamin C levels, through inactivation of NO signaling, are independent risk factors of CVD and related to increased mortality [32]. More specifically, it has also been demonstrated that a decrease in vitamin C levels was associated with carotid endothelial damage in patients with atherosclerosis, highlighting the positive role of vitamin C and antioxidants on NO levels [33]. The co-occurrence of periodontitis in CAD patients may be a possible pathway for the observed deterioration of endothelial function via decreased vitamin C levels. Periodontal treatment clinically decreased serum vitamin C and antioxidants levels in patients with chronic kidney disease [34].

As a support of the present study, several lines of evidence have shown that stimulating oxidative stress conditions, such as periodontitis and CAD, may have led to the lower the production of vitamin C, which in turn could augmen<sup>t</sup> serum and salivary CRP levels [35]. The high inflammation present during periodontitis and CAD is believed to accelerate vitamin C oxidation [35]. In accordance with our results, Amaliya et al. [36] found that low serum vitamin C and high CRP levels were associated in a dose-dependent manner in a sample of 98 subjects with periodontitis.

Moreover, while there are some observations on the serum vitamin C levels as a marker for endothelial dysfunction or CAD risk, there are no reports that analyze both salivary and serum vitamin C levels during periodontitis. However, the present study did not find a statistically significant correlation between serum and salivary vitamin C levels; salivary vitamin C levels were associated by hs-CRP levels. This could be explained by the fact that the saliva levels of vitamin C could mainly reflect the serum vitamin C levels or that the salivary vitamin C levels may have been influenced by the saliva collection method used in the present study [37].

In the present study, patients with CAD and with periodontitis plus CAD presented low salivary vitamin C levels, in accordance with previous studies which demonstrated that saliva contains many biochemical systems known to be involved in soft-tissue repair, and many antibacterial components, including lysozyme, lactoferrin, and salivary peroxidase [38]. Human whole saliva contains a complex peroxidase system, the major components of which include different forms of lactoperoxidase secreted by the salivary glands and myeloperoxidases from polymorphonucleocytes [39].

While the systemic impact of reduced vitamin C levels on endothelial dysfunction via decreased NO has been demonstrated, the effect of oral vitamin C is less clear. As a matter of fact, there are reports which show that periodontitis is positively associated with impaired salivary NO levels [19,24]. NO can be produced in the gingival tissues as part of the oral unspecific salivary antibacterial defense against anaerobic periodontopathogens bacteria [12,24,40]. In this regard, some reports showed high levels of NO synthesis and activity in the inflamed periodontal tissue [25,41–44]. Another explanation for the contradictory results may be due to the method of saliva collection. Moreover, it can also be argued that the difference in NO production at the periodontal level is probably different from NO in the bloodstream: In the mouth, it is an antibacterial defense, whereas systemically, it impacts endothelial function.

Moreover, endothelial dysfunctions in periodontitis patients with CAD could be due to a specific immunoreactive pathway in which vitamin C modulates an anti-inflammatory response against periodontopathic bacteria during periodontitis. It has been shown that vitamin C, during periodontitis is involved in immune response through the activated endothelium and its heat shock proteins that are present in the endothelium surface, finally stimulating some cross-reactive T-cells with particularity for host-activated antibodies [45,46]. This process, modulated by vitamin C, also affects the inducted defense mechanism mediated by NO, which promotes the hyperactivation of the endothelial cells that increases the risk of further infection or systemic inflammation due to periodontitis [47–49].

However, the present preliminary study presents some limitations. One of the main limitations is the cross-sectional nature of the study, which does not allow any evaluation on the impact of vitamin C levels on periodontitis, which should be assessed only with a longitudinal observation. Another limitation is the small sample size, which was due to matching age, gender, and education. An advantage of matching is the elimination of the impact of these confounding variables. Significant limitations also include the lack of analysis for dietary quality (e.g., intake of vitamin C and statins) and the analysis of alpha-tocopherol.
