Salivary glutathione peroxidase-1 levels in periodontitis

Abstract

BACKGROUND

Periodontitis consists of a chronic inflammatory disease of the periodontal tissue. In addition to inflammation, oxidation is also involved in periodontitis. Glutathione peroxidase (GPx) is an antioxidant enzyme. There are 8 forms of GPx (GPx 1-8) and GPx-1 is the most abundant. The comparison of salivary GPx-1 levels in patients with periodontitis and periodontally healthy subjects has been explored in small sample size studies (the largest including 101 subjects) and the results are contradictory.

AIM

To compare salivary GPx-1 concentrations in subjects with and without periodontitis in a study with a larger sample size. In addition, novel aims were exploring the possible association of salivary GPx-1 concentrations with periodontitis by a regression analysis and determining the capability of salivary GPx-1 concentrations to predict the diagnosis of periodontitis by a receiver operating characteristic (ROC) analysis.

METHODS

Observational and prospective study including subjects with and without periodontitis. Salivary samples were taken to determine GPx-1 concentrations. A multivariate logistic regression analysis was performed to determine whether salivary GPx-1 levels were independently associated with periodontitis. A ROC analysis was carried out to determine the capability for the diagnosis of periodontitis by salivary GPx-1 concentrations.

RESULTS

We found lower salivary GPx-1 concentrations in 57 subjects with periodontitis than in 86 without periodontitis. We found that salivary GPx-1 concentrations showed an area under the curve for periodontitis diagnosis of 76% (95%CI: 68%-83%; P < 0.001) in ROC analysis, and that salivary GPx-1 levels < 1214 pg/mL showed an independent association with periodontitis (odds ratio: 6.25; 95%CI: 2.844-13.717; P < 0.001) in regression analysis.

CONCLUSION

To our knowledge, our series has the largest sample size on salivary GPx-1 concentrations in periodontitis. Novel findings of our study were that salivary GPx-1 levels were able to diagnose periodontitis and were independently associated with periodontitis. However, our preliminary study has some limitations as there were significant differences in age between subjects with and without periodontitis, other antioxidant enzymes or total antioxidant capacity were not assessed, other biological samples were not analyzed, and GPx-1 genetic variations were not studied. Nevertheless, we think that the results of our study could encourage further research on the role of salivary biomarkers concentrations (as GPx-1) in the management of periodontitis.

Key Words: Glutathione peroxidase; Salivary; Periodontitis; Oxidation; Severity; Diagnosis

Core Tip: Periodontitis consists of a chronic inflammatory disease of periodontal tissue. In addition to inflammation, oxidation is also involved in periodontitis. Glutathione peroxidase (GPx)-1 is an antioxidant enzyme. Novel findings of our study were that salivary GPx-1 levels were able to diagnose periodontitis and were independently associated with periodontitis.

INTRODUCTION

Periodontitis consists of a chronic inflammatory disease of periodontal tissue, that has a high prevalence and could lead to many dental visits and loss of teeth[1]. In addition to inflammation, oxidation is also involved in periodontitis[2-6].

Free radicals are produced during cellular metabolism, have an unpaired electron and are very unstable molecules that need to steal electrons from other molecules to stabilize themselves[7-9]. There are different free radicals, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive sulfur species and reactive carbonyl species. Among ROS are hydroxyl radical (HO^•), hydroxide ion (HO⁻), singlet oxygen, superoxide anion, peroxide ion, hydrogen peroxide (H₂O₂) and nitric oxide (NO^•). Among RNS are NO^•, peroxynitrite, nitrogen dioxide and dinitrogen trioxide. In the process of free radicals’ stabilization, the attacked molecules (lipid, proteins, nucleic acids) are oxidized (due to donation of electrons to free radicals) and its structure and function deteriorates.

There are different endogenous and exogenous antioxidants that act to avoid or repair cellular damage caused by oxidation[10-13]. Antioxidants act in different ways to avoid oxidation, such as neutralizing free radicals by donating an electron to them, activating antioxidant enzymes that accelerate chemical reactions for neutralizing free radicals, blocking pro-oxidant enzymes that generate free radicals (for example, blocking nicotinamide adenine dinucleotide phosphate oxidase or NADPH oxidase, xanthine oxidase, lipoxygenases) and chelating metals (ferritin, lactoferrin, albumin, and ceruloplasmin) to inhibit ROS production dependent of metals (iron, copper). Among antioxidant enzymes are antioxidant enzymes as superoxide dismutase (SOD), thioredoxin reductase, peroxiredoxin, glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT). Among low molecular weight nonenzymatic oxidants are uric acid, bilirubin, melatonin and amino acids (tryptophan, methionine, histidine, lysine, cysteine, arginine and tyrosine). In addition to those endogenous antioxidants, there are exogenous antioxidants such as vitamins (A, C, and E) and trace elements (selenium, zinc, manganese, copper, iron).

The glutathione system involves glutathione tripeptide, antioxidant enzyme GPx and antioxidant enzyme GR[10-13]. Glutathione is a tripeptide that is made of amino acids glutamate, cysteine, and glycine. Glutathione has 2 forms, reduced glutathione form or glutathione sulfhydryl form (GSH) and oxidized glutathione form or glutathione disulfide form (GSSG). GSH has properties of nonenzymatic antioxidant by donating electrons to ROS, and not GSSG. GSH detoxifies ROS, but in this process GSH is oxidized and it transforms into GSSG.

GPx catalyzes detoxification of hydrogen peroxide by reducing free hydrogen peroxide to water (2 GSH + H₂O₂ → GSSG + 2 H₂O). There are 8 forms of GPx (GPx 1-8). GPx-1 is the most abundant, and it is a selenium-containing enzyme.

GR, also known as glutathione-disulfide reductase, catalyzes the reaction of reduction of GSSH to GSH transferring electrons from nicotinamide adenine dinucleotide phosphate (NADPH or reduced form of NADP), and subsequently NADP⁺ (oxidized form of NADP) and GSH appears (GSSG + NADPH → GSH + NADP⁺). Oxidative stress elevates GSSG levels, and afterwards the action of GR facilitates the conversion of GSSG into GSH to be able to neutralize ROS again.

ROS play a protective role in the periodontal tissue when there is a balance between ROS and antioxidant concentrations. ROS allows the killing of pathogens and promotes the response of the host immune system (with releasing of cytokines and attracting immune cells to destroy the microorganism pathogens). The equilibrium between ROS and antioxidant concentrations allows the maintaining homeostasis in the periodontal tissue structures. However, if this balance is broken due to higher ROS concentrations compared to antioxidant concentrations, then those higher ROS concentrations cause inflammation and damage in the periodontal tissues, and finally the destruction of the periodontal tissues. The homeostatic alveolar bone neo-apposition/bone resorption (with the balance of osteoclasts and osteoblasts) disappears and becomes pathologic bone resorption (due to a higher activity of osteoclasts in respect to osteoblasts)[2-6].

The presence of microorganism pathogens in the periodontal pocket stimulate the response of host immune cells (such asCD4 helper T-lymphocytes) and appears an inflammatory response (with the liberation of pro-inflammatory cytokines, such as interleukins and tumor necrosis factor-alpha) which attract immune cells as neutrophils and macrophages to the inflamed site. Those cells kill pathogens with their capacity of phagocytosis (to engulf the pathogens) and of ROS liberation (to destroy the pathogens). Additionally, physiological levels of ROS are vital for preserving the function of periodontal ligament stem cells, which are essential in periodontal regeneration and tissue repair. However, when the infection persists, the balance between ROS and antioxidants is broken and the ROS increase can damage proteins, DNA, and lipids, and produce periodontal tissues damage and alveolar bone resorption[2-6].

ROS could be dangerous due to the direct effects on periodontal tissues and due to indirect effects by activating different pathways. In the presence of an oxidative stress environment, there is an increase of degradation and a reduction of production of different elements of the periodontium such as proteins (collagen, elastin) and glycosaminoglycans (hyaluronic acid), and therefore the destruction of periodontal tissue[2-6].

Increased ROS concentrations in the periodontal pockets can upregulate signaling of different pathways that lead to the aggravation of periodontal state. Elevated ROS produces the liberation of the nuclear factor-κB which causes the expression of pro-inflammatory cytokines, chemokines, matrix metalloproteinases. All those produce the activation of inflammasomes (complex of multiple proteins in the cytoplasm) that consist of NOD-like receptor protein 3, pro-caspase-1 and apoptosis-associated speck-like protein containing a CARD (CARD is a caspase recruitment domain). And those promotes apoptosis of periodontal fibroblast and causes damage to the periodontal ligament[2-6].

In addition, ROS also could induce apoptosis of periodontal fibroblast through the activation of mitochondrial apoptosis pathway liberating apoptosis-inducing factor and liberating cytochrome-C (which will produce caspase-9 activation)[2-6].

Besides, ROS also induced activation of mitogen-activated protein kinases (MAPKs), such p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N terminal kinase, and extracellular signal-related kinases, and all those favors apoptosis of periodontal fibroblasts (by activation of caspase-3, increasing Bax levels and decreasing Bcl-2 expression)[2-6].

Increased ROS causes the overproduction of cytokines that break the axis between the receptor activator of nuclear factor kappa-B ligand, which is an activator of osteoclastogenesis, and the osteoprotegerin, also named osteoclastogenesis inhibitory factor or also tumour necrosis factor receptor superfamily member 11B, which is an inhibitor of osteoclastogenesis; inducing osteoclastic activity leading to alveolar bone destruction[2-6].

Therefore, we hypothesized that subjects with higher salivary GPx, GR and GSH concentrations could have higher capacity to neutralize ROS and lower risk to develop periodontitis and its severe forms. However, subjects with lower salivary concentrations of GPx, GR and GSH could have lower capacity to neutralize ROS and higher risk to develop periodontitis and its severe forms.

The comparison of salivary GPx-1 levels in patients with periodontitis and periodontally healthy subjects has been explored in studies with small sample sizes and the results are contradictory[14-25], and the study with the largest sample size with 101 subjects was the study by Kluknavská et al[19]. It was found higher salivary GPx-1 levels in patients with periodontitis in some studies[14-18], higher salivary GPx-1 levels in periodontally healthy subjects in other studies[19-22], and no differences in those levels between patients with periodontitis and periodontally healthy subjects in other studies[23-25]. In one meta-analysis by Mohideen et al[7] published in 2024, including the most of those studies[14-24], were found higher salivary GPx-1 levels in periodontally healthy subjects than in patients with periodontitis.

However, the possible association of salivary GPx-1 concentrations with periodontitis by a regression analysis has not been explored, nor the ability of salivary GPx-1 concentrations to diagnose periodontitis by a receiver operating characteristic (ROC) analysis. Thus, we conducted this study with the aim of comparing salivary GPx-1 concentrations in subjects with and without periodontitis in a larger sample size study. In addition, novel aims were exploring the possible association of salivary GPx-1 concentrations with periodontitis by a regression analysis and determining the capability of salivary GPx-1 concentrations to predict the diagnosis of periodontitis by a ROC analysis.

MATERIALS AND METHODS

Design and subjects

This was a prospective and observational study. It was carried after the approval by the Clinical Research Ethics Committee of the Hospital Universitario de Canarias (CHUC_2023_138; 30 November 2023). Clínica Dental Cándido (from La Laguna, Tenerife, Canary Islands, Spain) recruited the subjects and salivary samples. The subjects must sign an informed consent before their participation in the study.

We included subjects with and without periodontitis. The group of subjects with periodontitis included subjects with loss of periodontal tissue or bone. The group of subjects without periodontitis included subjects that had periodontal health (with nonexistence of bleeding on probing or only in less than 10% of locations) or localized gingivitis (with the existence of bleeding in 10%-30% of sites). Current criteria for the diagnosis and severity classification of periodontitis (stages I to IV) were used[26]. We excluded subjects younger than 18 years.

Variables recorded

We registered, in addition to age and sex, the consumption of tea, alcohol, tobacco, coffee and drugs, and the dental hygiene. Besides, we registered the personal history of systemic lupus erythematosus, rheumatoid arthritis, obesity (when body mass index ≥ 30 kg/m²), diabetes mellitus, hypercholesterolemia, oral cancer, cardiovascular disease and arterial hypertension. We also recorded the treatment with radiotherapy, methotrexate dosed for rheumatoid arthritis or immunosuppressive therapy.

Salivary samples

The samples were taken in the morning (8 am to 10 am) to try reducing the influence of circadian rhythm on the concentration of salivary biomarker. We used the technique of Navazesh to recollect samples of not stimulated saliva[27]. The samples were centrifuged for 10 minutes at 3000 rpm, and afterwards the supernatant was distributed in Eppendorf tubes and were located at -80 °C to determination of biomarker concentration.

Previously, due to our research line of periodontitis, we determined salivary concentration of uric acid[28], malondialdehyde[29], and thioredoxin-1[30]. Thus, some subjects were already included in some of those publications.

Analysis of GPx-1 concentrations

GPx-1 concentrations in saliva were evaluated using a solid-phase Enzyme Linked Immunosorbent Assay (Invitrogen, Thermo Fisher Scientific Inc., Whaltam, MA, United States), according to the manufacturer’s protocol. Briefly, in each appropriated well: 100 μL standards (for standard curve) and 100 μL saliva (unknown samples) were incubated for 90 minutes at 37 °C. Thoroughly aspirate the solution (not washing). Add 100 μL of Biotinylated Detection Ab working solution and incubated 60 minutes at 37 °C. After washing each well (3 times) with wash buffer, was added 100 μL HRP Conjugate Working Solution into each well and incubate for 30 minutes at 37 °C. After washing each well (5 times) with wash buffer, 90 μL of Substrate Reagent (3,3′,5,5′-Tetramethylbenzidine or TMB as chromogenic substrate) were added to each well and incubate 15 minutes at 37 °C avoiding direct light. Finally, 50 μL of Stop Solution (1 M H₂SO₄) was added each well (yellow colour) at room temperature. The kit uses a calibration curve with a range 0-2000 pg/mL. Samples and standards absorbance units were read in a microplate spectrophotometer at 450 nm (Spectra MAX-190, Molecular Devices, Sunnyvale, CA, United States). A two-phase exponential decay provided the best standard curve fit (the correlation coefficients R² of different kits was ranged between 0.9975 and 0.9973). The limit of detection of the different assays was established in 9.6 pg/mL. Intra-assay and inter-assay coefficients of variation were calculated at 4.17% and 5.15%, respectively.

Statistical analysis

We used χ² test for the comparison of number and percentage of events in the categorical variables, and Mann-Whitney U-test for the comparison of median and percentiles 25-75 in the continuous variables. Spearman’s rho coefficient was used for the study of correlation between different continuous variables. To determine whether differences in salivary GPx-1 levels according to other variables were statistically significant, Bonferroni correction for multiple comparisons was applied.

A multivariate logistic regression analysis was carried out to determine whether some variables (including salivary GPx-1 concentrations) were associated with periodontitis, introducing those variables showing P value ≤ 0.05 when comparing subject groups and showing reasonable number of events.

A ROC analysis was carried out to explore the capability of periodontitis diagnosis by salivary GPx-1 concentrations, and the area under curve (AUC) was reported. Besides, specificity, sensitivity, negative and positive predictive values (negative predictive value and positive predictive value), and negative and positive likelihood ratios (LR- and LR+) for the cut-off of salivary GPx-1 concentrations < 1214 pg/mL. The cut-off of salivary GPx-1 was selected on the basis of Youden[31] index.

We found statistically significant differences in age between subjects with and without periodontitis. Thus, we have repeated the same analyses after matched (for age and sex) the subjects with and without periodontitis. The only variable that showed differences statistically significant between subjects with and without were salivary GPx-1 levels; however, in the regression analysis were included, besides of salivary GPx-1 levels, also body mass index and never smoker. Those variables were included due that showed the lowest P values in the comparison of salivary GPx-1 levels and showed a reasonable number of events.

Statistical analyses were made with the programs MedCalc Statistical Software version 22.016 (MedCalc Software Ltd, Ostend, Belgium) and SPSS version 17.0 (SPSS Inc., Chicago, IL, United States).

RESULTS

Results in the total cohort (n = 143)

We included 143 subjects (57 with periodontitis and 86 without it). The number of subjects and the salivary GPx-1 concentrations for each subject group according to its periodontal state are shown in Table 1, and significant differences in salivary GPx-1 concentrations were found between subject groups (P < 0.001). In addition, we found a negative correlation between salivary GPx-1 concentrations and periodontitis severity (rho = -0.46; P < 0.001).

Table 1 Number of subjects and salivary glutathione peroxidase-1 concentration in each periodontal stage, n (%).

	Total (n = 143)	Salivary GPx-1 (pg/mL), median (P25-P75)	P value
			< 0.001
Periodontal health	86 (60.1)	1511 (887-2024)
Periodontitis stage I	16 (11.2)	1154 (358-1333)
Periodontitis stage II	23 (16.1)	978 (645-1457)
Periodontitis stage III	13 (9.1)	629 (292-1128)
Periodontitis stage IV	5 (3.5)	435 (62-768)

GPx-1: Glutathione peroxidase-1.

The group of subjects with periodontitis had lower salivary GPx-1 concentrations (P < 0.001), were older (P < 0.001), had higher rate of cardiovascular disease (P = 0.02) and of arterial hypertension (P < 0.001), and had lower rate of never smoker history (P < 0.001) and of tea consumption (P = 0.001) than the group of subjects without periodontitis. We found no significant differences between the groups of subjects with and without periodontitis in sex, diabetes mellitus, body mass index, alcohol, coffee, rheumatoid arthritis, hypercholesterolemia, radiotherapy, obesity and immunosuppressive therapy (Table 2). All subjects reported that they practice daily oral hygiene with tooth brushing and toothpaste. None of the subjects had a history of systemic lupus erythematosus, nor oral cancer.

Table 2 Comparisons between subjects without and with periodontitis, n (%).

	Subjects without periodontitis (n = 86)	Subjects with periodontitis (n = 57)	P value
Gender female	64 (74.4)	36 (63.2)	0.19
Age (years) - median (P25-P75)	35 (20-43)	59 (51-68)	< 0.001
Arterial hypertension	4 (4.7)	17 (29.8)	< 0.001
Cardiovascular disease	0	4 (7.0)	0.02
Hypercholesterolemia	2 (2.3)	2 (3.5)	0.99
Diabetes mellitus	0	3 (5.3)	0.06
Rheumatoid arthritis	1 (1.2)	3 (5.3)	0.30
Metrotexate for rheumatoid arthritis	1 (1.2)	0	0.99
Immunosupressive therapy	1 (1.2)	1 (1.8)	0.99
Radiotherapy	0	1 (1.8)	0.40
Body mass index (kg/m2) - median (P25-P75)	24.6 (22.4-26.6)	24.8 (22.5-27.6)	0.66
Obesity	11 (12.8)	8 (14.0)	0.99
Never smoker	68 (79.1)	25 (43.9)	< 0.001
Coffee	66 (76.7)	50 (87.7)	0.13
Tea	22 (25.6)	3 (5.3)	0.001
Alcohol	34 (39.5)	30 (52.6)	0.17
Salivary GPx-1 levels (pg/mL) - median (P25-P75)	1511 (887-2024)	924 (324-1252)	< 0.001
Salivary GPx-1 levels < 1214 pg/mL	26 (30.2)	42 (73.7)	< 0.001

GPx-1: Glutathione peroxidase-1.

We have found that salivary GPx-1 concentrations were influenced by smoking (P = 0.002). However, after Bonferroni correction for multiple comparisons, no significant differences were found in salivary GPx-1 levels according to age (P = 0.01), cardiovascular disease (P = 0.04), arterial hypertension (P = 0.03), diabetes mellitus (P = 0.06), hypercholesterolemia (P = 0.61), sex (P = 0.87), rheumatoid arthritis (P = 0.51), immunosuppressive therapy (P = 0.65), obesity (P = 0.97), coffee (P = 0.77), tea (P = 0.40), alcohol (P = 0.73), and radiotherapy (P = 0.63).

Salivary GPx-1 concentrations < 1214 pg/mL [odds ratio (OR): 6.25; 95%CI: 2.844-13.717; P < 0.001] and age (years) (OR: 1.10; 95%CI: 1.062-1.134; P < 0.001) were the variables associated with periodontitis according to multiple logistic regression analysis (Table 3).

Table 3 Multiple logistic regression analysis to determine that factors are associated with periodontitis.

	Odds ratio	95%CI	P value
Age (years)	1.10	1.062-1.134	< 0.001
Salivary GPx-1 levels < 1214 pg/mL	6.25	2.844-13.717	< 0.001
Never smoker (yes vs non)	0.84	0.371-1.882	0.66
Arterial hypertension (yes vs non)	1.60	0.650-3.927	0.31
Tea consumption (yes vs non)	0.56	0.133-2.342	0.43
Gender (female vs male)	0.84	0.374-1.873	0.67

GPx-1: Glutathione peroxidase-1.

AUC of salivary GPx-1 concentrations for diagnosis of periodontitis was of 76% (95%CI: 68%-83%; P < 0.001) (Figure 1). The cut-off salivary GPx-1 concentrations < 1214 pg/mL for diagnosis of periodontitis had positive predictive value 62% (53%-70%), sensitivity of 74% (60%-85%), positive likelihood ratio 2.4 (1.7-3.5), negative predictive value 80% (72%-86%), specificity 70% (59%-79%). and negative likelihood ratio 0.4 (0.2-0.6).

Figure 1 Receiver operating characteristic analysis to determine the capability of salivary glutathione peroxidase-1 concentrations to periodontitis diagnosis. AUC: Area under curve; CI: Confidence interval.

Results in the subjects matched for age and sex (n = 60)

When we compared 30 subjects without periodontitis and 30 with periodontitis matched for age and sex, remain significant differences in salivary GPx-1 concentrations between subject groups (P = 0.01) (Table 4) and a correlation between salivary GPx-1 concentrations and periodontitis severity (rho = -0.44; P < 0.001).

Table 4 Number of subjects and salivary glutathione peroxidase-1 concentration in each periodontal stage, n (%) in the subjects matched for age and sex.

	Total (n = 60)	Salivary GPx-1 (pg/mL), median (P25-75)	P value
			0.01
Periodontal health	30 (50.0)	1471 (856-2009)
Periodontitis stage I	12 (20.0)	1188 (359-1490)
Periodontitis stage II	12 (20.0)	1096 (372-1463)
Periodontitis stage III	5 (8.3)	304 (164-1128)
Periodontitis stage IV	1 (1.7)	44 (-)

GPx-1: Glutathione peroxidase-1.

The group of subjects with periodontitis had higher salivary GPx-1 concentrations than subjects without periodontitis (P = 0.002). We found no other statistically significant differences between the groups of subjects with and without periodontitis in other variables (Table 5).

Table 5 Comparisons between subjects without and with periodontitis, n (%) in the subjects matched for age and sex.

	Subjects without periodontitis (n = 30)	Subjects with periodontitis (n = 30)	P value
Gender female	20 (66.7)	20 (66.7)	0.99
Age (years) - median (P25-P75)	52 (44-61)	51 (46-58)	0.83
Arterial hypertension	4 (13.3)	5 (16.7)	0.99
Cardiovascular disease	0	1 (3.3)	0.99
Hypercholesterolemia	2 (6.7)	0	0.49
Diabetes mellitus	0	0	0.99
Rheumatoid arthritis	0	1 (3.3)	0.99
Metrotexate for rheumatoid arthritis	0	0	0.99
Immunosupressive therapy	0	0	0.99
Radiotherapy	0	0	0.99
Body mass index (kg/m2) - median (P25-P75)	24.6 (23.3-27.1)	24.9 (23.0-27.9)	0.91
Obesity	4 (13.3)	5 (16.7)	0.99
Never smoker	20 (66.7)	18 (60.0)	0.79
Coffee	28 (93.3)	25 (83.3)	0.42
Tea	4 (13.3)	3 (10.0)	0.99
Alcohols	12 (40.0)	14 (46.7)	0.80
Salivary GPx-1 levels (pg/mL) - median (P25-P75)	1471 (856-2009)	1092 (297-1399)	0.002
Salivary GPx-1 levels < 1214 pg/mL	9 (30.0)	19 (63.3)	0.02

GPx-1: Glutathione peroxidase-1.

After to apply Bonferroni correction for multiple comparisons, there were no significant differences in salivary GPx-1 concentrations according to sex (P = 0.86), age (P = 0.34), arterial hypertension (P = 0.96), cardiovascular disease (P = 0.18), hypercholesterolemia (P = 0.21), rheumatoid arthritis (P = 0.84), body mass index (P = 0.18), obesity (P = 0.34), never smoker (P = 0.18), coffee (P = 0.88), tea (P = 0.86), and alcohol (P = 0.47).

We found in the multiple logistic regression analysis that the only variable independently associated with periodontitis was salivary GPx-1 concentration < 1214 pgl/mL (OR: 4.55; 95%CI: 1.413-14.623; P = 0.01) (Table 6).

Table 6 Multiple logistic regression analysis to determine that factors are associated with periodontitis in the subjects matched for age and sex.

	Odds ratio	95%CI	P value
Salivary GPx-1 levels < 1214 pg/mL	4.55	1.413-14.623	0.01
Never smoker (yes vs non)	1.09	0.340-3.478	0.89
Body mass index (kg/m2)	1.05	0.917-1.190	0.51

GPx-1: Glutathione peroxidase-1.

Salivary GPx-1 concentrations had an AUC for diagnosis of periodontitis of 73% (95%CI: 60%-86%; P = 0.002).

DISCUSSION

To our knowledge, our series has the largest sample size on salivary GPx-1 concentrations in periodontitis. We found that subjects with periodontitis showed lower salivary GPx-1 concentrations than subjects without periodontitis. In addition, we found a negative correlation between salivary GPx-1 concentrations and periodontitis severity. Novel findings of our study were that salivary GPx-1 levels were able to diagnose periodontitis and were independently associated with periodontitis.

The comparison of salivary GPx-1 levels in patients with periodontitis and periodontally healthy subjects has been explored in small sample size studies[14-25], and the study with the largest sample size with 101 subjects was the study by Kluknavská et al[19]. Thus, to our knowledge, our series has the largest sample size (n = 143) on salivary GPx-1 concentrations in periodontitis.

The results of studies assessing salivary GPx-1 levels in patients with periodontitis are contradictory[14-25], reporting higher salivary GPx-1 levels in patients with periodontitis in some studies[14-18], higher salivary GPx-1 levels in periodontally healthy subjects in other studies[19-22], and no differences between patients with periodontitis and periodontally healthy subjects in other studies[23-25]. One meta-analysis including the most of those studies[14-24] published in 2024 by Mohideen et al[7] concluded that patients with periodontitis showed lower salivary GPx-1 levels. Thus, the results of our study are in agreement with those of the meta-analysis by Mohideen et al[7].

A novel finding of our study was that low salivary GPx-1 concentrations were associated independently of other factors with periodontitis according to the results of multivariate logistic regression analysis.

Another novel finding of our study was that salivary GPx-1 concentrations could help in the diagnosis of periodontitis according to the results of receiver operating characteristic analysis. However, the AUC in our study (76%) was not high[32], and salivary GPx-1 concentrations could not be used as a single parameter for periodontitis diagnosis.

In one study there was found a negative correlation between salivary GPx-1 concentrations and periodontitis severity[21], and in other study the contrary (a positive correlation)[33]. In the study by Naresh et al[21] with 90 subjects (60 with periodontitis and 30 without periodontitis) was found a negative correlation with 4 clinical periodontal parameters (plaque index, gingival index, probing pocket depth and clinical attachment level). In the study by Novaković et al[33] with 21 subjects with periodontitis (without healthy control subjects) was found a positive correlation between salivary GPx-1 concentrations and plaque index; but not with other clinical periodontal parameters (gingival index, bleeding on probing, probing pocket depth and clinical attachment loss). Thus, our findings in respect of the negative association between salivary GPx-1 concentrations and periodontitis severity are in agreement with one of those studies[21] and are contrary with the other[33].

We found that besides salivary GPx-1 levels, another factor associated with periodontitis was the age as it was also previously reported[34]. However, other risk factors of periodontitis described in other studies as rheumatoid arthritis[35], sex[36], diabetes mellitus[37], obesity[38], systemic lupus erythematosus[39], consumption of drugs, oral cancer, dental hygiene, alcohol, immunosuppression, coffee, tea and tobacco[40] and arterial hypertension[41] were not found to be associated with periodontitis in our series. This may be related to sample size.

A promising fact is that GPx-1 activity has been increased by different agents in rat models of periodontitis, and in addition it has been accompanied by reduced alveolar bone loss[42-44]. In a study published by Sezgin et al[42], it was found that the oral administration of resveratrol in rats with periodontitis increased gingival GPx activity and reduced alveolar bone loss. In a study published by Zhu et al[43], it was found that the administration of MIL-47(V)-F, which is a nanozyme that mimics the function of GPx, in rats with periodontitis reduced ROS formation and reduced alveolar bone loss. In a study published by Keskin et al[44], it was found that the oral administration of rutin (vitamin P) in rats with periodontitis increased gingival GPx activity, increased total oxidant status, reduced total oxidant status, and reduced alveolar bone loss.

We must recognize that our study had some limitations. First, GPx-1 concentrations were only determined in saliva and not in other biologic samples. GPx-1 has been found in higher concentrations in gingival tissue than in saliva samples[18]. We used saliva samples, which are easier and less invasive; however, salivary levels might not perfectly reflect systemic and tissue concentrations. Gingival tissue samples can provide stronger results in terms of diagnostic accuracy and scientific specificity compared to saliva samples. Second, we have not assessed other antioxidant enzymes such as SOD and CAT, or total antioxidant capacity. Third, we did not consider the early or late stage of periodontitis, and it is possible that the low salivary GPx-1 levels in these patients suggest that they are chronic stage. Fourth, we did not estimate a sample size; however, our study had a sufficient sample size to find in regression analysis that salivary GPx-1 concentrations were independently associated with periodontitis. In addition, we have found in a post hoc analysis that the study had greater than 80%. Fifth, we have not assessed the association between GPx-1 genetic variations and salivary GPx-1 concentrations. In several studies, it has been found an association between GPx-1 genetic variations, plasma GPx-1 activity and different non-oral diseases[45-47]. Specifically in the oral diseases, the association between GPx-1 genetic variations and different diseases has been scarcely studied[48,49], its association with the disease studied was not found, and the GPx-1 activity was not studied. It was not found an association of GPx-1 genetic variations with the susceptibility to caries[48] and with the development of oral cancer[49]. The most common among these genetic variations in GPx is in the single nucleotide polymorphism rs1050450 in the GPx-1 gene. It produces the apparition of proline (Pro) or leucine (Leu) at codon 198 (Pro198 Leu)[50]. In one study it was found that the expression of distinct GPx-1 alleles (T or C) affects its subcellular localization (mitochondria or cytoplasm) and its function. The authors suggested that it is plausible that the sequestration of GPx-1 in the mitochondria makes it less efficient at reducing hydrogen peroxide[50]. Sixth, there were significant differences in age between subjects with and without periodontits; however, we have matched the subjects for age and sex. The number of subjects that we could matched was 60 of the 143, and the results were similar. Seventh, other confounding factors that we have not included in the regression analysis due to sample size of our study or that we have not registered could have influenced our findings.

On the contrary, we think that the novel findings of our study about salivary GPx-1 levels were able to diagnose periodontitis and were independently associated with periodontitis were the strength of our study. In addition, those findings were similar when the subjects with and without periodontitis were matched for age and sex. Salivary sample collection is a non-invasive and easy sampling procedure. Thus, we think that the results of our study and all those of previous studies could motivate more research about the role of salivary biomarkers concentrations on the management of periodontitis.

Further research directions could be the following: Investigate other antioxidant enzymes or oxidation biomarkers in saliva or other samples (periodontal tissues) in periodontitis. Exploring the potential of GPx-1 as a therapeutic target of periodontitis in humans (if the administration of agents that increase GPx-1 activity could reduce oral oxidative stress and improve periodontal health). Analyze the potential influence of GPx-1 genetic variations in salivary GPx-1 concentrations, salivary oxidative stress and the development of periodontitis and their severe forms. Explore the possibility that the determination of salivary biomarkers could help in the early prediction of periodontal disease and of their severe forms of disease (before the development of clinical and radiographic symptoms).

CONCLUSION

To our knowledge, our series has the largest sample size on salivary GPx-1 concentrations in periodontitis. Novel findings of our study were that salivary GPx-1 levels were able to diagnose periodontitis and were independently associated with periodontitis. However, our preliminary study has some limitations as there were significant differences in age between subjects with and without periodontits, other antioxidant enzymes or total antioxidant capacity were not assessed, other biological samples were not analyzed, and GPx-1 genetic variations were not studied. Nevertheless, we think that the results of our study could encourage further research on the role of salivary biomarkers concentrations (as GPx-1) in the management of periodontitis.

ACKNOWLEDGEMENTS

Centro Integrado de Formacion Profesional Los Gladiolos (Santa Cruz de Tenerife, Spain) has facilitated the inclusion of participants in the study.