A Comparative Evaluation of Serum and Salivary Antioxidant Levels in Healthy Controls, Gingivitis, and Periodontitis Patients

ABSTRACT

Objective:

Periodontitis is a chronic inflammatory disorder that gives rise to tissue damage and loss due to the complex interaction between pathogenic bacteria and the host’s immune response. The aim of this study is to determine the lipid peroxidation, total antioxidant (AO) capacity (TAC), and superoxide dismutase (SOD) levels in patients diagnosed with chronic gingivitis and chronic periodontitis and in periodontally healthy control subjects

Methods:

The subjects were divided into three groups. Blood sample and saliva were collected. TAC, MDA, and SOD levels were estimated by spectrophotometric methods. SOD in the saliva samples was analyzed by using the enzyme-linked immunosorbent assay (ELISA) kit method.

Results:

The mean age of the study population in the control group was 45.21 ± 13.12, which in the gingivitis group was 44.12 ± 9.10 and in the periodontitis group was 42.19 ± 12.11. TAC and SOD levels were significantly reduced in both serum and saliva in the periodontitis compared to control and the gingivitis group. MDA levels were significantly increased in the serum and the saliva of the periodontitis compared to control and the gingivitis group.

Conclusion:

Oxidative stress, or the imbalance between oxidants and AO, is a major factor in the development and course of periodontitis.

INTRODUCTION

Periodontitis is a chronic, multifactorial inflammatory disease of the teeth’s supporting tissues that causes progressive destruction of the periodontal ligament and alveolar bone, resulting in clinical attachment loss, periodontal pocket formation, gingival recession, and, if left untreated, tooth loss. However, this defense mechanism unwittingly aids in the destruction of the periodontium. As periodontitis advances, it causes continuing attachment loss, alveolar bone resorption, and, in extreme cases, tooth loss^.[1] The primary etiologic agents such as gram-negative anaerobic or facultative bacteria, which cause tissue damage, are present within the subgingival biofilm,[2] but periodontal breakdown occurs due to inappropriate host response to these pathogens.[3] Once bacteria present within the periodontium enter the blood via circulation, an inflammatory and immune response occurs. This creates a host–microbial interaction involving cellular and humoral factors.[2] The immune response that reacts against the bacteria can be extracellular or intracellular, which activates both T cells and B cells. The primary defense mediators against bacterial pathogens[4] are neutrophils and polymorphonuclear leukocytes (PMNs), and they generate increased levels of reactive oxygen species (ROS), which can cause tissue damage in the bacterial pathogen pathway directly or the host–microbial interaction pathway.[5]

During radical oxygen production, there is an increase in levels of reactive oxygen species (ROS) which includes both oxygen free radicals and other nonradical oxygen derivatives. These include superoxide (O₂), hydroxyl (OH), hydroperoxyl (HOO), nitric oxide (NO), alkoxy (RO), singlet oxygen, ozone (O₃), hypochlorous acid (HOCl), and hydrogen peroxide (H₂O₂).[6] In normal physiology, there is a dynamic equilibrium between ROS activity and antioxidant (AO) defense capacity, and, when that equilibrium shifts in favor of ROS, it results in oxidative stress (OS) by either a reduction in AO defense or an increase in ROS production or activity.[7] Due to the prolonged exposure of increased concentrations of ROS, oxidative damage generally occurs. Likewise, when there is a decrease in AO defense against ROS, damage can also occur.[8] Intracellularly, it can cause direct damage to the cell structure and membrane, resulting in dysfunction and cell death, while extracellularly, it can cause direct damage to the connective tissue and extracellular matrices.

Polyunsaturated fatty acids are mainly seen in cell membranes and lipoproteins. When ROS react with these fatty acids, an uncontrolled lipid peroxidation (LPO) occurs. These lipid peroxides break down to certain cytotoxic aldehydes and less toxic aldehydes (e.g., malondialdehyde (MDA)). These aldehydes increase in concentration due to OS, leading to periodontal destruction and deoxyribonucleic acid (DNA) damage. MDA is one of the low-molecular weight, mutagenic and carcinogenic end products of LPO and prostaglandin biosynthesis. It is often measured as an index of peroxidation [2]. It exists in free state as well as in the bound state. Most of the studies suggest that the MDA levels are increased in periodontal disease due to OS. Saliva is used as a potential biomarker for prognosis and diagnosis of many systemic and oral diseases. Saliva can be used as a biomarker to assess MDA levels since it is easy to collect and is a noninvasive and convenient approach toward patients,[9] unlike GCF which is difficult to collect and is time-consuming.

Strong AO defense mechanisms, including superoxide dismutases (SOD), catalase, glutathione peroxidase, uric acid, ascorbic acid, glutathione, lipoic acid, carotenoids, vitamin E, and ubiquinol, are present in cells, tissues, and bodily fluids to prevent oxidative damage.[10] When ROS production and AO defenses are out of balance, OS takes place in tissues. The cumulative effect of the primarily nonenzymatic AO found in plasma and bodily fluids is reflected by the total antioxidant capacity (TAC), an integrated metric.[11] It is hypothesized that TAC measurement could reveal details about the equilibrium between AO and oxidant systems.

Periodontitis is characterized by high OS, which alters the equilibrium of ROS and AO defenses, resulting in gradual tissue damage. Saliva is a simple, noninvasive diagnostic medium; therefore, comparing salivary and serum markers may indicate its reliability for clinical assessment. This research is, therefore, required to identify significant oxidative indicators that can enable early detection and better management of periodontal disease. Thus, by evaluating and correlating the serum and salivary MDA, SOD and TAC levels were estimated in patients diagnosed with chronic gingivitis and chronic periodontitis and in periodontally healthy control subjects.

MATERIALS AND METHODS

ETHICAL CLEARANCE

The present study was approved by the ethics committee of AB Shetty Memorial Institute of Dental Science, Nitte (deemed to be University; ABSM/EC/111/2017). Informed consent was taken from all the patients before sample collection.

SAMPLE SIZE CALCULATION

The sample size was calculated using the formula,[12]

Zα-1.96 at 95% confidence interval

Zβ- 0.84 at 80% power

σ - 0.15, pooled standard deviation

0.11 mean difference

STUDY POPULATION

Ninety subjects were recruited from the Department of Periodontics, AB Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangalore. The study subjects were divided into three groups, i.e., group I, group II, and group III.

Group I (Control group): 30 subjects with healthy gingiva.

Group II (Gingivitis group): 30 subjects with gingivitis.

Group III (Periodontitis group): 30 subjects with periodontitis.

SCREENING INCLUDES THE FOLLOWING:

• Medical and dental history of the subjects were collected.

• Periodontal examination.

• Clinical attachment levels (CAL) were determined with a UNC-15 probe.

• Gingival index was recorded according to the criteria given by Loe and Silness, 1963.[13]

ASSESSMENT OF CLINICAL PARAMETERS

ASSESSMENT OF CLINICAL PARAMETERS SUCH AS GINGIVAL INDEX WAS PERFORMED ACCORDING TO THE METHOD DESCRIBED BY LOE AND SILNESS[10]

0 – normal gingiva.

1 – mild inflammation, slight change in color, and slight edema; no bleeding on probing.

2 – moderate inflammation, redness, edema, and glazing; bleeding on probing.

3 – severe inflammation, marked redness and edema, and ulceration; tendency to spontaneously bleed.

CLINICAL ATTACHMENT LEVELS

CAL is the distance from the cemento-enamel junction (CEJ) to the base of the gingival sulcus or pocket.

SALIVA COLLECTION

The technique used for collecting unstimulated whole saliva (3 mL) was the spitting method. The collected saliva was centrifuged at 3000 g at room temperature for 15 min. This fraction was then aliquoted into storage vials and stored at -80°C until further analysis.

SERUM COLLECTION

A volume of 5 mL of blood was collected in sterile tubes and centrifuged to separate the serum, which was stored at −80°C until further analysis.

ESTIMATION OF TOTAL ANTIOXIDANTS

The assay relies on the principle of conversion of molybdenum (Mo VI) by reducing agents like AO to molybdenum (Mo V), which further reacts with phosphate under acidic pH, resulting in the formation of a green complex, the intensity of which can be read spectrophotometrically at 695 nm. A volume of 100 µL of the sample (serum/saliva) was pipetted into a clean test tube, and 5% TCA was added to precipitate out the proteins in the sample. The mixture was allowed to stand for 5 min and centrifuged. A volume of 100 µL of the clear supernatant was transferred into a clean test tube, and 1 mL of TAC reagent was added to it; the mixture was incubated in a water bath at 90°C for 90 min. Simultaneously, a blank is also maintained by substituting 100 µL of water instead of the sample in the reaction mixture. It was then cooled, and optical density was read at 695 nm against the blank. The concentration of the total AO in the serum/saliva was obtained by plotting the standard graph, and the concentration of TAC was expressed as µg/dL.

ESTIMATION OF MDA CONCENTRATION

MDA concentration in the serum/saliva was measured by thiobarbituric acid—reactive substances (TBAARS) assay according to Kei (1978). Here, TBA reacts with MDA to form a pink complex, which can be spectrophotometrically read at 532 nm. The concentration of the MDA in the serum/saliva was obtained by plotting the standard graph, and the concentration of MDA was expressed as µM/L.

SPECTROPHOTOMETRIC ANALYSIS FOR SUPEROXIDE DISMUTASE

The substrate used for the assay consists of nitro blue tetrazolium chloride (NBT) which reacts with superoxide anions produced upon illumination of riboflavin in the presence of methionine as an electron donor to produce formazan, a blue complex. The SOD present in the sample will act on the superoxide anions produced by riboflavin and thereby reduce the net superoxide anions in the substrate, leading to decreased production of formazan manifested by the decreased intensity of the blue color formed. The decrease in the formation of formazan is directly proportional to the amount of SOD in the sample; a 50% decrease in the formation of formazan is taken as one unit of SOD. Briefly, 500 µL of heparinized blood was centrifuged at 1800 rpm for 10 min. The upper plasma layer was separated, and 500 µL of normal saline was added to the erythrocyte layer, mixed well, and centrifuged. The upper layer was discarded, and fresh normal saline was added to the erythrocytes; this step was repeated for two more times to wash the erythrocytes. The control for each sample analyzed has to be maintained. Common standard and blanks for each set of illumination are maintained. A volume of 100 µL of red blood corpuscles (RBC) lysates is diluted further by the addition of 400 µL of 0.05 M phosphate buffer to get a final erythrocyte dilution of 1:20.

• i.Test: 0.3 mL riboflavin, 2.5 mL methionine, 0.1 mL NBT, and 0.1 mL RBC lysate.
• ii.Control: 2.5 mL methionine, 0.3 mL riboflavin, and 0.1 mL 0.05 M phosphate buffer.

• 0.1 mL RBC lysate.

• iii.Standard: 2.5 mL methionine, 0.3 mL riboflavin, and 0.1 mL NBT,

• 0.1 mL 0.05 M phosphate buffer.

• iv.Blank: 2.5 mL methionine, 0.3 mL riboflavin, and 0.2 mL 0.05 M phosphate buffer

Following illumination, immediately, the optical density of all the reaction mixtures was read at 560 nm. The units of the enzyme present in the sample were calculated using the following formula and expressed as U/mg Hb.

SOD present/mg Hb = SOD activity/Hb/20 Dilution factor = 20

SOD activity*20 SOD activity/mg Hb = ———————Hb

= ——————— U/mg Hb.

Salivary SOD levels were estimated by using the ELISA kit method and expressed as U/µL.

STATISTICAL ANALYSIS

Statistical analyses were performed using the following software tool: Statistical Package for the Social Sciences (version 16.2 for Windows; SPSS Inc, Chicago, Illinois, USA). Data were expressed as mean ± standard deviation. Multiple comparisons were done by analysis of variance. For all statistical analyses, P <0.05 was considered statistically significant.

RESULTS

The mean age of the study population in the control group was 45.21 ± 13.12, which in the gingivitis group was 44.12 ± 9.10 and in the chronic periodontitis group was 42.19 ± 12.11. Clinical parameters of the different groups are shown in Table 1.

Table 1.

Clinical parameters of the study population

	Group I	Group II	Group III
Mean GI (Gingival Index)	0.75 ± 0.03	2.22 ± 0.6	2.18 ± 0.41
Mean Probing Pocket Depth (PPD) (mm)	1.8 ± 0.04	2.1 ± 0.5	4.96 ± 0.07
Mean CAL (mm)	–	–	5.75 ± 0.5

CAL = clinical attachment levels

TAC was found to be significantly reduced in both serum and saliva in the periodontitis group (ug/dL; 60.04 ± 11.18 and 12.10 ± 12.01) compared to control (152.77 ± 47.32, 25.13 ± 17.01) and the gingivitis group (90.19 ± 13.61, 15.11 ± 10.17). MDA levels were significantly increased in the serum and the saliva of the periodontitis group (μM/L; 1.790 ± 0.88, 1.782 ± 0.45) compared to control (0.846 ± 0.46, 0.485 ± 0.29) and the gingivitis group (1.19 ± 0.45, 0.528 ± 0.30). SOD levels were significantly reduced in both serum and saliva in the periodontitis group (444.13 ± 66.18 U/mg Hb and 0.094 ± 0.01 U/µL) compared to control (1149.2 ± 29.62 U/mg Hb, 0.323 ± 0.15 U/µL) and the gingivitis group (820.85 ± 34.18 U/mg Hb, 0.213 ± 2.14 U/µL) [Table 2].

Table 2.

Biochemical analysis of MDA, TAC, and SOD in serum and saliva

Parameters	Group I		Group II		Group III		P value
	Serum	Saliva	Serum	Saliva	Serum	Saliva
TAC (μg/dL)	152.77 ± 47.32	25.13 ± 17.01	90.19 ± 13.61	15.11 ± 10.17	60.04 ± 11.18	12.10 ± 12.01	<0.001*
MDA (μM/L)	0.846 ± 0.46	0.485 ± 0.29	1.19 ± 0.45	0.528 ± 0.30	1.790 ± 0.88	1.782 ± 0.45	<0.001*
SOD	1149.2 ± 29.62 U/mg Hb	0.323 ± 0.15 (U/µL	820.85 ± 34.18 U/mg Hb	0.213 ± 2.14 U/µL	444.13 ± 20.18 U/mg Hb	0.094 ± 0.01 U/µL	<0.001*

TAC = total antioxidant capacity, SOD = superoxide dismutase

^*P <0.001 is considered statistically significant

CAL and TAC in serum and saliva across the three groups had a positive correlation. CAL loss decreases from periodontal health to gingivitis and periodontitis, suggesting a positive (not significant) relationship between AO defense and periodontal tissue degradation.

A positive correlation (not significant) was observed between CAL and MDA levels in serum and saliva in the periodontitis group. As clinical attachment loss progresses from periodontal health to gingivitis and periodontitis, MDA levels increase steadily, indicating increased LPO and OS with disease severity [Tables 3 and 4].

Table 3.

Correlation between CAL and antioxidant property

Parameters	Group III		Spearman correlation
	Serum	Saliva	Serum	Saliva
TAC (μg/dL)	60.04 ± 11.18	12.10 ± 12.01	0.137	0.007
MDA (μM/L)	1.790 ± 0.88	1.782 ± 0.45	0.161	0.014
SOD	444.13 ± 20.18 U/mg Hb	0.094 ± 0.01 U/µL	−0.142	−0.219

CAL = clinical attachment levels, TAC = total antioxidant capacity, SOD = superoxide dismutase

Table 4.

Correlation between PPD and antioxidant property

Parameters	Group I Spearman correlation		Group II Spearman correlation		Group III Spearman correlation
	Serum	Saliva	Serum	Saliva	Serum	Saliva
TAC (μg/dL)	˗0.044	0.082	0.052	0.246	0.068	−0.021
MDA (μM/L)	0.083	0.054	−0.161	0.166	0.167	0.048
SOD	0.026	−0.173	0.037	−0.134	0.002	0.016

TAC = total antioxidant capacity, SOD = superoxide dismutase

Spearman’s correlation reveals a negative correlation (not significant) between CAL and SOD levels in serum and saliva in periodontitis. As clinical attachment loss increases from periodontal health to gingivitis and periodontitis, SOD levels gradually drop, indicating a decrease in AO defense as periodontal tissue deterioration occurs.

PPD and total AO capacity in serum and saliva across the three groups had a positive correlation (not significant), except for TAC in the serum of Group I and saliva of Group III. Periodontal inflammation and OS rise in parallel with PPD, which may lead to a compensatory overexpression of AO defenses.

A positive correlation (not significant) was observed between PPD and MDA levels in serum and saliva in periodontitis, except for serum in Group II, which was negatively correlated. Deeper pockets of periodontitis contain more pathogenic biofilms as PPD rises, which results in increased neutrophil activity, excessive production of ROS, and elevated MDA levels.

Spearman’s correlation reveals a negative correlation (not significant) between PPD and SOD levels in serum and saliva in all three groups, except for saliva of groups I and II. A rise in PPD is indicative of increasing inflammation, which may result in SOD activity inhibition or consumption and decreased enzymatic efficiency as a result of an ongoing oxidative load.

DISCUSSION

Gingivitis and periodontitis are characterized by gum inflammation, which includes redness, swelling, and textural changes. Periodontal pockets form because of structural deterioration in periodontitis. The disease is often painless, with discomfort only occurring during acute flare-ups, which are usually caused by periodontal abscess formation or decreased tooth support. As a result, the problem frequently goes undetected until it reaches an advanced state, delaying treatment.[14] The prevalence of periodontal disease, particularly its mild and moderate forms, is over 50% in adult populations worldwide, whereas the prevalence of its severe form rises, particularly in the third and fourth decades of life, globally at about 10%.[15,16] This research is, therefore, required to identify significant oxidative indicators that can enable early detection and better management of periodontal disease.

The mean age of the study population in the control group was 45.21 ± 13.12; in the gingivitis group, it was 44.12 ± 9.10; and in the periodontitis group, it was 42.19 ± 12.11. Total AO capacity was found to be significantly reduced in both serum and saliva in the periodontitis (60.04 ± 11.18 ug/dL and 12.10 ± 12.01ug/dl) compared to control (152.77 ± 47.32 ug/dL and 25.13 ± 17.01 ug/dL) and the gingivitis group (90.19 ± 13.61 ug/dL and 15.11 ± 10.17 ug/dL). MDA levels were significantly increased in the serum and the saliva of the periodontitis (1.790 ± 0.88 μM/L and 1.782 ± 0.45 μM/L) compared to control (0.846 ± 0.46 μM/L and 0.485 ± 0.29 μM/L) and the gingivitis group (1.19 ± 0.45 μM/L and 0.528 ± 0.30 μM/L). A study found that the patients with chronic periodontitis had higher levels of OS in both serum and saliva. These patients’ serum and saliva have a slightly reduced TAC. In these patients, elevated MDA levels without alterations in AO status may lead to both local and systemic problems.[17]

Neutrophils are the most common inflammatory cells that accumulate in periodontal tissue and gingival sulcus following the induction of host defensive responses by periodontal pathogenic bacteria in biofilms. Neutrophils are believed to be the primary source of ROS in periodontitis.[18] In addition to acting as a second messenger, modulating biological processes, and providing cytoprotective advantages, ROS have the capacity to physiologically eliminate periodontal infections. On the other hand, an excess of ROS can cause tissue degradation and an antagonistic cycle between ROS and the immuno-inflammatory cascade. Through their DNA, pathogenic bacteria and lipopolysaccharides present in subgingival dental plaque activate TNF-alpha and other Toll-like receptors.[19] Hyper-responsive PMNs generate ROS in response to inflammatory cytokines. Conversely, activating proteins-1 and NF-kβ increase matrix metalloproteinase concentrations and activate osteoclasts, both of which cause tissue damage. When periodontal tissue is damaged, an several oxidized proteins, inflammatory mediators, and lipid peroxides are created. These products further activate neutrophils, fibroblasts, and macrophages, increasing the generation of ROS.[20]

SOD levels were significantly reduced in both serum and saliva in the periodontitis (444.13 ± 66.18 U/mg Hb and 0.094 ± 0.01 U/µL) compared to control (1149.2 ± 29.62 U/mg Hb and 0.323 ± 0.15 U/µL) and the gingivitis group (820.85 ± 34.18 U/mg Hb and 0.213 ± 2.14 U/µL). In contrary to our results, a study found that people with periodontal disease may have higher salivary SOD levels as a defense mechanism against OS brought on by the condition.[21] The use of distinct assay principles for assessing SOD in serum (spectrophotometric riboflavin-NBT technique) and saliva (ELISA) prevents the direct comparability of enzymatic activity between the two fluids because the methods measure different analytical endpoints. This methodological mismatch should be recognized as a significant limitation of the study, reflecting limits linked to sample matrix properties and assay sensitivity^.[22] Because thiobarbituric acid combines with a number of different aldehydes and biomolecules, causing nonspecific chromogen production and an overestimation of MDA levels, the TBARS assay has limited specificity for MDA. Furthermore, TBARS readings should be considered an indication of LPO rather than a precise measure of MDA concentration because the harsh acidic and high-temperature conditions of the assay might lead to artifactual MDA formation.[23]

Thus, OS plays a major role in the pathogenesis of periodontal disease, which highlights the importance of targeted treatments such AO supplements and lifestyle changes to reduce its detrimental effects. Clinicians may be able to better treat and prevent periodontal disease by addressing OS. Ultimately, this will improve the dental health and quality of life of those impacted.[24]

CAL assessment using manual probes has a number of drawbacks, such as operator-dependent variability, inconsistent probing force, and variations in angulation, all of which can impact measurement accuracy and repeatability. Furthermore, measurement mistakes and decreased sensitivity in detecting subtle changes in CAL might result from factors such as gingival inflammation, patient discomfort, probe tip diameter, and difficulties recognizing the CEJ, particularly in longitudinal investigations.[25]

CONCLUSION

From the present study, we observed an increase in salivary MDA level in the chronic periodontitis group compared to gingivitis and healthy control groups and significantly decreased TAC and SOD levels. OS, or the imbalance between oxidants and AO, is a major factor in the development and course of periodontitis. The imbalance between tissue loss and gain in periodontal disease can be caused by a number of factors, including as an aggressive infection, uncontrolled chronic inflammation, impaired healing, or all of the above at once. Therefore, a better understanding of the various aspects of the disease at the individual level and the development of customized treatment modalities, such as immunotherapies and inflammatory modulators, are necessary for effective disease management.

CONFLICTS OF INTEREST

There are no conflicts of interest.

ETHICAL POLICY AND INSTITUTIONAL REVIEW BOARD STATEMENT

The present study was approved by the ethics committee of AB Shetty Memorial Institute of Dental Science, Nitte (deemed to be University; ABSM/EC/111/2017).

PATIENT DECLARATION OF CONSENT

Informed consent was taken from all the patients before sample collection.

AUTHOR CONTRIBUTIONS

Not applicable.

DATA AVAILABILITY STATEMENT

Not applicable.

List of abbreviations

• AO Antioxidant

• CAL Clinical attachment levels

• CEJ Cemento-enamel junction

• DNA Deoxyribonucleic acid

• ELISA Enzyme-linked immunosorbent assay

• LPO Lipid peroxidation

• MDA Malondialdehyde

• NBT Nitro blue tetrazolium chloride

• OS Oxidative stress

• PMNs Polymorphonuclear leukocytes

• PPD Probing Pocket Depth

• RBC Red Blood Corpuscles

• ROS Reactive oxygen species

• SOD Superoxide dismutase

• SPSS Statistical Package for the Social Sciences

• TAC Total antioxidant capacity

• TCA Trichloroacetic Acid

Funding Statement

Nil.