Low salivary uric acid levels are independently associated with periodontitis

Abstract

BACKGROUND

Lower salivary uric acid concentrations, the most abundant antioxidant agent in saliva, have been observed in patients with periodontitis compared to individuals with periodontal health. However, the independent association of salivary uric acid levels with periodontitis, accounting for other risk factors, as well as its association with periodontitis severity, has not been investigated.

AIM

To the independent association of salivary uric acid levels with periodontitis and the association of salivary uric acid levels with the severity of periodontitis.

METHODS

This observational and prospective study measured salivary uric acid levels in subjects with periodontitis (characterized by periodontal loss of tissue) and in subjects without periodontitis (periodontal health or localized gingivitis in < 30% of sites). Multivariate regression analysis was performed to determine independent factors associated with periodontitis. Spearman’s rho correlation coefficient was used to assess the relationship between salivary uric acid levels and periodontitis severity. A receiver operating characteristic analysis was carried out to evaluate the diagnostic performance of salivary uric acid levels in periodontitis, reporting the area under curve (AUC) and its 95%CI.

RESULTS

We included 121 subjects, 61 of them with periodontitis and 60 without periodontitis (39 with periodontal health and 21 with local gingivitis). Subjects with periodontitis compared to those without periodontitis were older (P < 0.001), had higher salivary uric acid levels (P = 0.002), higher rate of arterial hypertension history (P = 0.001) and higher rate of never-smoker history (P < 0.001). The AUC for periodontitis diagnosis by salivary uric acid levels was 66% (95%CI: 57%-75%; P < 0.001) and the better cut-off point was 111 nmol/mL. Multiple logistic regression analysis showed an independent association of salivary uric acid levels < 111 nmol/mL (OR = 6.14; 95%CI: 2.015-18.721; P = 0.001) with periodontitis after controlling for age, never-smoker history and arterial hypertension. A negative correlation of salivary uric acid levels and periodontitis severity was observed (rho = -0.32; P < 0.001).

CONCLUSION

The two novel findings of our research were, first, that low salivary uric acid concentrations are independently associated with periodontitis, even after accounting for established risk factors. Second, salivary uric acid levels show a negative correlation with periodontitis severity.

Key Words: Oxidation; Salivary; Uric acid; Periodontitis; Gingivitis

Core Tip: Lower salivary uric acid concentrations, the most abundant antioxidant agent in saliva, have been observed in patients with periodontitis compared to individuals with periodontal health. The two novel findings of our research were, first, that low salivary uric acid concentrations are independently associated with periodontitis, even after accounting for established risk factors. Second, salivary uric acid levels show a negative correlation with periodontitis severity.

INTRODUCTION

Periodontitis is a chronic inflammatory disease characterized by microorganism invasion of the periodontium. It has a high prevalence, affecting 50% of individuals in its mild forms and 7% in advanced stages and has important economic consequences, with an estimated $79 billion annually worldwide[1].

Several periodontitis risk factors have been described, such as diabetes mellitus[2], systemic lupus erythematosus and rheumatoid arthritis[3], sex[4], arterial hypertension[5], age[6], obesity[7], oral cancer, dental hygiene, immunosuppression and consumption of toxic substances (drugs, tobacco, tea, alcohol, coffee)[8].

In periodontitis, the destruction of the periodontium occurs due to the activation of pathophysiological pathways such inflammation[9,10], oxidation[11-14] and programmed cell[15,16].

Free radicals, highly reactive compounds, can cause damage to nucleic acids, proteins and lipids. Different types of free radicals are involved in periodontitis[11-14]: (1) Reactive nitrogen species (nitric oxide, nitrite and nitrate) that cause nitrosative stress; and (2) Reactive oxygen species (ROS) (hydroxyl radical, peroxyl radical and superoxide anion radical) that cause oxidative stress.

Different types of antioxidant agents exist to prevent damage from free radicals which can be categorized into enzymatic and non-enzymatic antioxidant defense system[11-14]. Oxidative damage will depend on the balance of free radical production and antioxidant systems activity. The antioxidant enzyme system includes glutathione peroxidase, superoxide dismutase, thioredoxin, catalase and glutathione reductase. The antioxidant non-enzymatic system includes albumin, melatonin, glutathione, vitamin E, bilirubin, vitamin C and uric acid. Uric acid accounts for approximately 70% of the total antioxidative capacity of saliva[17,18].

Two meta-analyses have analyzed the relationship between saliva and blood concentrations of uric acid and chronic periodontitis[19,20]. Both concluded that patients with chronic periodontitis compared to healthy subjects had lower uric acid saliva concentrations and higher concentrations of uric acid in blood. In the meta-analysis published by Uppin et al[19] in 2022 8 studies were included with 159 periodontitis patients and 149 controls, and in the meta-analysis published by Ye et al[20] in 2023 9 studies with 233 periodontitis patients and 174 controls were included. However, the association of salivary uric acid levels with periodontitis, after adjusting for risk factors, and its association with severity of periodontitis have not been reported. Therefore, the novel objectives of our study were to determine whether these associations exist.

MATERIALS AND METHODS

Design and subjects

This study was initiated after obtaining approval from the Clinical Research Ethics Committee of the Hospital Universitario de Canarias. Prior to inclusion, all participants provided written informed consent. This prospective and observational study was conducted at Clínica Dental Cándido (La Laguna, Tenerife, Canary Islands, Spain). Patients with periodontitis (charachterized by periodontal tissue loss) and subjects without periodontitis (including those with localized gingivitis in < 30% sites and those with periodontal health) were included. Diagnosis of periodontitis and classification of its severity were based on current internationally accepted criteria[21]. Subjects younger than 18 years were excluded.

Definitions

Clinical periodontal health was established when the subject showed no bleeding on probing or showed bleeding in less than 10% of locations and showed no clinical interproximal attachment loss or bone loss. Localized gingivitis was established when the subject showed bleeding in 10%-30% of locations, and didn’t present clinical interproximal attachment loss or bone loss. Periodontitis was established when the subject showed bone loss or clinical interproximal attachment loss. Periodontitis severity classification was assessed using the following 3 criteria: (1) Clinical interproximal attachment loss: Stage I with < 3 mm, Stage II with 3 a 4 mm, Stage III or IV with ≥ 5 mm; (2) Radiographic bone loss: Stage I with coronal third < 15%, Stage II with coronal third 15%-33%, Stage III or IV with middle or apical third; (3) Tooth loss: Stage I or II with none, Stage III with ≤ 4 teeth, Stage III with ≥ 5 teeth.

Variables recorded

We recorded the following variables: Age, salivary uric acid levels, body mass index (BMI) (kg/m²), sex, arterial hypertension, diabetes mellitus, obesity (BMI≥ 30 kg/m²), never smoker, coffee, tea, alcohol, rheumatoid arthritis, immunesupressive therapy, radiotherapy, methotrexate, drugs consumption, oral cancer, systemic lupus erythematosus, chemotherapy and dental hygiene with toothbrushing.

Salivary samples

We used the Navazesh technique to obtain whole unstimulated saliva samples[22]. Saliva collection took place in the morning (from 8 to 10 a.m.) to reduce possible influences by circadian rhythm on salivary biomarker concentrations. Participants did not drink, smoke, or brush their teeth for at least two hours prior to sample collection. At the Dental Clinic, subjects rinsed their mouths with 10 mL. of deionized water 3 times. They then sat comfortably for 30 minutes with their heads slightly tilted forward, eyes open, and avoiding oro-facial movements. During this period, they refrained from swallowing saliva, allowing it to accumulate in the mouth before expelling it into a collection bottle. Saliva samples were centrifuged for 10 minutes at 3000 rpm at 24 °C to remove debris and cells. The supernatants were then pipetted into Eppendorf tubes and stored at -80ºC for subsequent salivary concentration determinations. Some subjects included in this study had also been included in a previous publication of our team, in which salivary nitrite levels were reported[23]. In the present study, salivary uric acid levels were assessed.

Salivary uric acid level analysis

Salivary uric acid levels were measured using the Randox kit reagents (Randox Laboratories, Country Antrim, United Kingdom), based on the method developed by Fossati et al[24]. Uric acid is converted by uricase into allantoin and H₂O₂, which, under the catalytic influence of peroxidase, oxidizes 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-amonophenazonen to form a red-violet quinoneimine compound. For the assay, 0.02 mL of saliva was combined with 0.3 mL of enzyme reagent. The red-violet compound reaction developed over 15 minutes at room temperature. Each sample was then relocated to a 96-well plate and read at 515 nm (λ_max) by means of a microplate spectrophotometer reader (Spectra MAX-190, Molecular Devices, Sunnyvale, CA, United States). The assay's detection limit was established at 9.45 nmol/mL. Intra-assay and inter-assay coefficients of variation were calculated at 1.68% and 2.75%, respectively.

Statistical analysis

We used Kruskal Wallis test to determine whether had significant differences existed between subject groups in salivary uric acid concentrations. Continuous variables were compared between subject groups using the Mann-Whitney U-test, Mann-Whitney U-test, while categorical variables were analyzed using the χ² test. We analyzed the association between salivary uric acid and periodontitis severity using Spearman’s rho correlation coefficient. We carried out a receiver operating characteristic analysis to evaluate the diagnostic performance of salivary uric acid levels for periodontitis, reporting the area under curve (AUC) and its 95%CI. We selected the point of salivary uric acid levels < 111 nmol/mL based on the Youden[25]. We performed a multivariate regression analysis to determine whether an independent association between salivary uric acid levels and periodontitis exists controlling for other periodontitis risk factors. In the regression analysis we included those variables that showed a P value ≤ 0.05 in the comparison of subjects with or without periodontitis (salivary uric acid levels, age, never smoker status and arterial hypertension). Statistical analyses were caried out using the following statistical programs: SPSS version 17.0 program (SPSS Inc., Chicago, IL, United States), MedCalc Statistical Software version 22.016 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org; 2023), and LogXact-4 for Windows version 4.1. (Cytel Software Corporation).

RESULTS

A total of 121 subjects were included, 61 with periodontitis and 60 without periodontitis (39 with periodontal health and 21 with local gingivitis). The number of subjects in each periodontal state and their salivary uric acid concentrations are showed in Table 1. We found statistically significant differences between subject groups in salivary uric acid concentrations (P = 0.01).

Table 1 Description of periodontal state in all subjects of the study, n (%)/median (25^th-75^th percentiles).

	Total (n = 119)	Subjects without periodontitis (n = 60)	Subjects with periodontitis (n = 61)	Uric acid (nmol/mL)	P value
					0.01
Periodontal health	39 (32.2)	39 (65.0)	0	126 (79-203)
Localized gingivitis	21 (17.4)	21 (35.0)	0	112 (86-142)
Periodontitis stage I	17 (14.0)	0	17 (27.9)	102 (53-137)
Periodontitis stage II	24 (19.8)	0	24 (39.3)	93 (62-171)
Periodontitis stage III	13 (10.7)	0	13 (21.3)	86 (53-111)
Periodontitis stage IV	7 (5.8)	0	7 (11.5)	61 (21-94)

Subjects with periodontitis compared to those without periodontitis were older (P < 0.001), had higher salivary uric acid levels (P = 0.002), higher rate of arterial hypertension history (P = 0.001) and higher rate of never-smoker history (P < 0.001). In our series, no subjects had with a history of drug consumption, oral cancer, systemic lupus erythematosus, chemotherapy or dental hygiene without toothbrushing. Statistically significant differences were not found in BMI, sex, diabetes mellitus, obesity, coffee, tea, alcohol, rheumatoid arthritis, immunosupressive therapy, radiotherapy and metrotexate between subjects suffering or not periodontitis (Table 2).

Table 2 Comparisons between subjects with and without periodontitis, n (%)/median (25^th-75^th percentiles).

	Subjects without periodontitis (n = 60)	Subjects with periodontitis (n = 61)	P value
Age (years)	41 (34-49)	60 (52-68)	< 0.001
Salivary uric acid (nmol/mL)	112 (82-176)	89 (54-119)	0.002
Body mass index (kg/m²)	24.8 (22.9-26.7)	24.8 (22.6-28.4)	0.81
Sex female	40 (66.7)	38 (62.3)	0.71
Arterial hypertension	3 (5.0)	19 (31.1)	0.001
Diabetes mellitus	0	5 (8.2)	0.06
Obesity	7 (11.9)	9 (14.8)	0.79
Never smoker	47 (78.3)	26 (42.6)	< 0.001
Coffee	51 (85.0)	54 (88.5)	0.60
Tea	5 (8.3)	3 (4.9)	0.49
Alcohol	27 (45.0)	31 (50.8)	0.59
Rheumatoid arthritis	1 (1.7)	4 (6.6)	0.37
Immunosupressive therapy	1 (1.7)	3 (4.9)	0.62
Radiotherapy	0	3 (4.9)	0.24
Metrotexate	1 (1.7)	0	0.50

The AUC for periodontitis diagnosis by salivary uric acid levels was 66% (95%CI: 57%-75%; P < 0.001) and the better cut-off point was 111 nmol/mL (Figure 1). Multiple logistic regression analysis showed an independent association of salivary uric acid levels < 111 nmol/mL (OR = 6.14; 95%CI: 2.015-18.721; P = 0.001) with periodontitis after controlling for age, never-smoker history and arterial hypertension (Table 3). A negative correlation of salivary uric acid levels and periodontitis severity was observed (rho = -0.32; P < 0.001).

Figure 1  Receiver operating characteristic analysis using salivary uric acid levels for diagnostic of periodontitis.

Table 3 Multiple logistic regression analyses to determine factors associated with periodontitis.

	Odds ratio	95%CI	P value
Salivary uric acid levels < 111 nmol/mL (yes vs non)	6.14	2.015-18.721	0.001
Age (years)	1.13	1.073-1.197	< 0.001
Never smoker (yes vs non)	0.44	0.154-1.254	0.12
Arterial hypertension (yes vs non)	1.49	0.289-7.661	0.63

DISCUSSION

We found that subjects with periodontitis, compared to those without periodontitis, had lower salivary uric acid levels, which is consistent with the findings of two previous meta-analyses[19,20]. In addition, a novel finding of our study was the independent association between low salivary uric acid concentrations and periodontitis after adjusting for other risk factors. Another novel and interesting result was the negative association between salivary uric acid levels and the severity of periodontitis.

We also identified two other variables independently associated with periodontitis: Never-smoking history and age. These findings are in agreement with the results of previous studies[6,8]. However, no other variables were associated with periodontitis in our series, and the relatively low sample size may have influenced this outcome.

Conflicting results have been reported regarding uric acid concentrations in saliva and blood in patients with and without periodontitis. Two meta-analyses concluded that patients with periodontitis, compared to those with periodontal health, had lower salivary concentrations and higher blood concentrations of uric acid[19,20]. Several potential explanations exist for the lower salivary uric acid concentrations observed in patients with periodontitis. One possibility is increased consumption of salivary uric acid due to elevated oxidative stress during periodontal infection, where uric acid can be oxidized by ROS into allantoin. Another explanation is that salivary uric acid may serve as a substrate for microorganism synthesis in dental plaque[26]. However, the exact mechanisms remain unclear, and further investigation is needed.

We acknowledge that our study had some limitations. A larger sample size would have allowed us to include only those with periodontal health in the group of subjects without periodontitis, excluding those with localized gingivitis. Additionally, we did not determine uric acid levels in other samples (gingival crevicular fluid or blood). We did not calculate the sample size because it was incidental, aiming to explore an issue that had not been previously addressed. However, the sample size was sufficient to report the association of salivary uric acid concentrations with periodontitis and its severity. We excluded subjects under 18 years of age because they are often excluded in research studies. However, it could also be interesting to study these aspects in this population.

We believe that the most important point of our study is not that salivary uric acid levels can help in diagnosing periodontitis but that they could influence its development and severity. The diagnosis and classification of periodontitis severity are straightforward using clinical periodontal examination and X-rays, eliminating the need for saliva extraction. However, determining salivary uric acid levels could help assess the antioxidant status of saliva in patients with periodontitis and suggest the use of antioxidant mouth creams (e.g., with uric acid) for those with severe periodontitis. Logically, for this step, studies should first be conducted with mouth creams in animal models of periodontitis and subsequently in human patients.

Another important aspect could be whether the deviation of salivary uric acid levels in the mouths of patients with gingivitis and normal patients could predict the likelihood of their subsequent development of periodontitis in the future, and this could be another potential line of investigation.

Our modest study only attempts to put a small stone in the knowledge of periodontitis reporting for the first time an independent association between salivary uric acid levels and periodontitis and an association between salivary uric acid levels and severity of periodontitis.

CONCLUSION

In conclusion, the two novel findings of our research were the independent association of low salivary uric acid concentrations and periodontitis, regardless of other risk factors, and the negative association between salivary uric acid levels and periodontitis severity. Second, salivary uric acid levels show a negative correlation with periodontitis severity.

ACKNOWLEDGEMENTS

Clínica Dental Cándido (Laguna. Tenerife. Spain) clinic has paid the costs of consumables and reagents for the determinations.