Assessment of Levels of Plasma Oxidative Stress in Patient Having Aggressive Periodontitis before and after Full Mouth Disinfection.

AIM: The primary purpose of the study was to evaluate the levels of oxidative stress in plasma in patients with aggressive periodontitis (AgP) before and after full-mouth disinfection.
MATERIALS AND METHODS: Twenty-five healthy controls and 25 participants with aggressive periodontal were assessed for plaque index, probing pocket depth, papillary bleeding index, and clinical attachment level. Periodontal bone support was assessed by taking full mouth periapical radiographs. Full-mouth disinfection of the patient was done within 24 h of clinical assessment of AgP. These parameters were assessed at the baseline and after 8 weeks of initial periodontal therapy. Plasma samples were taken and evaluated for various oxidative stress markers.
RESULTS: Strong positive correlation was observed among periodontal parameters and levels of enzymatic/nonenzymatic biomarkers for oxidative stress (thiobarbituric acid-reactive substances [TBARS], glutathione peroxidase [GPX], and catalase [CAT]) (P < 0.05), before and after periodontal management. The patients with AgP had high levels of TBARS, GPX, and CAT levels in the plasma matched to the healthy individuals (P < 0.05).
CONCLUSION: Enzymatic and nonenzymatic oxidative stress may have a role in the pathogenesis AP. Initial periodontal treatment can lead to the reduction of these stresses. Copyright:

INTRODUCTION

Periodontitis is an immune-mediated inflammatory process, resulting in loss of periodontal attachment to the root surface and destruction of adjacent alveolar bone, which results in loss of tooth.[1] Aggressive periodontitis (AgP) is a multifactorial periodontal disease affecting the younger individual caused by the interaction of microbiologic, immunologic, genetic, and environmental/behavioral risk factors. These factors decide the onset, course, and severity of the disease.[2]

The main reason of periodontal tissue destruction is an inappropriate host response to microorganisms and their products. Polymorphonuclear leukocytes, the first line of defense, kill the microorganism by the oxidative and nonoxidative process. Oxidative killing (superoxide, hydroxyl radicals, hypochlorous acid, hydrogen peroxide, and chloramines) results in generation of reactive oxygen species (ROS), whereas nonoxidative killing is mediated by several lysosomal enzymes, peptides, and proteins.[3] In the case of AgP, neutrophils become hyperactive and result in excessive oxidative stresses and damage to the tissue. Thus, an imbalance between the proteolytic enzymes and their inhibitors and ROS and the antioxidant defense systems leads to the periodontal tissue destruction.[4]

In research studies, biomarkers serve a major role in measuring the disease progression. Enzymes, antioxidants, and the oxidation products of protein, lipids, and DNA are widely used as biomarkers of oxidative stress.[5] Very few studies have reported the biomarker levels of oxidative stress in the plasma of periodontitis patients.[6] In this study, biomarkers of lipid peroxidation, glutathione peroxidase (GPX), and catalase (CAT) were used for assessing antioxidant levels, and thiobarbituric acid-reactive substances (TBARS) were used for quantifying ROS damage in patients with AgP before and after full-mouth disinfection.

MATERIALS AND METHODS

Informed consent was obtained from the participants. The institutional ethical committee approved the study. Fifty participants aged 20–40 years were divided into two groups. Group I (n = 25 study group) consists of patients diagnosed with AgP. Group II (n = 25 control group) consists of healthy age- and sex-matched controls.

Inclusion criteria

Participants living under the same geographic and climatic condition with an almost similar lifestyle, food habits, and oral hygiene habits were included in the study.

Exclusion criteria

Participants with any other systemic disorders or on antibiotics or anti-inflammatory medication, who underwent periodontal treatment in the past 6 months and with smoking or alcohol habit or going through pregnancy/lactation/menopause were excluded from the study.

All participants were periodontally examined by measuring plaque index (PI),[7] probing pocket depth (PPD),[8] papillary bleeding index (PBI),[9] and clinical attachment level (CAL).[10] Periodontal bone support was assessed by taking full mouth periapical radiographs. CALs were recorded at six sites (mesial, median, and distal points at buccal and palatal aspects) using Williams periodontal probe of the teeth. The same investigator did all clinical measurements. Periodontal therapy consisted of oral hygiene instructions, followed by the full-mouth disinfection of the patient. Scaling and root planning with chlorhexidine irrigation was done within 24 h of clinical assessment of patients with aggressive periodontits.[11] These parameters were assessed at the baseline and after 8 weeks of initial periodontal therapy.

Collection of samples

Five milliliters fasting blood samples were withdrawn at the baseline, before periodontal therapy, and 4 weeks after initial periodontal treatment from the antecubital vein. For the estimation of GSH additional, 0.5 mL of the whole blood sample was added to tubes containing 0.5 mL of 10% TCA. The samples were then centrifuged at a speed of 3500 rpm for 10 min to separate plasma and transferred into cryogenic vials and stored in liquid nitrogen at −80°C until the assay.

Biochemical assays

The level of TBARS was assayed colorimetrically using the method described by Ohkawa et al.[12] at 530–535 nm in the plasma. CAT was measured colorimetrically[13] at 570 nm in the plasma. GPX was assayed by the Rotruck et al.[14] method and was read at 420 nm.

Statistical analysis

The result was expressed as the mean ± standard deviation. The comparison of different parameters and observations of assays between different groups was made by an independent student's t-test analysis (P < 0.05).

RESULTS

Table 1 shows a statistically significant difference between the periodontal parameters of the two groups. The PI and gingival status of Group I were deteriorated when compared to Group II. The values for PPD and CAL were 6.97 ± 0.42* mm and 7.01 ± 0.21* mm in Group I as compared to normal values in the control group.

Table 1

Comparison of periodontal parameters between the study and control group

Groups	Mean±SEM
	PI	PBI	PPD	CAL
Case (Group I)	3.01±0.14*	60.12±0.92*	6.97±0.42*	7.01±0.21*
Control (Group II)	0.21±0.02	6.29±0.35	1.24±0.42	1.54±0.43

*P<0.05 significant. SEM: Standard error of mean, PI: Plaque index, PBI: Papillary bleeding index, PPD: Probing pocket depth, CAL: Clinical attachment level

Table 2 shows the comparison of the clinical parameter before and after initial therapy in the study group. The statistically significant reduction was noted; probing depth and gain in attachment were noticed after periodontal treatment. The plaque score was 3.01 ± 0.14* before treatment and 1.45 ± 0.32 after treatment. PBI and PPD were 60.12 ± 0.92* and 6.97 ± 0.42 mm before and 34.15 ± 0.24 and 5.05 ± 1.23 mm, respectively, after treatment. Similarly, the CAL was 7.01 ± 0.21* mm and 4.70 ± 1.46 mm before and after treatment.

Table 2

Comparison of periodontal parameters before and after treatment within the study group

Case	Mean±SEM
	PI	PBI	PPD	CAL
Before treatment	3.01±0.14*	60.12±0.92*	6.97±0.42*	7.01±0.21*
After treatment	1.45±0.32	34.15±0.24	5.05±1.23	4.70±1.46

*P<0.05 significant. SEM: Standard error of mean, PI: Plaque index, PBI: Papillary bleeding index, PPD: Probing pocket depth, CAL: Clinical attachment level

Table 3 depicts the comparison between the levels of oxidative stress (enzymatic and nonenzymatic) between the two groups. In Group I, the initial level of TBARS was 9.98 ± 1.42* nmol/mg, and in the Group II, it was 4.65 ± 0.87 nmol/mg. The levels of GPX and CAT followed the same trends, i.e., they were raised significantly in the study group.

Table 3

Comparison of antioxidant levels between the study and control group

Groups	Mean±SEM
	TBARS	GPX	CAT
Case	9.98±1.42*	187.44±22.75*	151.64±20.54*
Control	4.65±0.87	119.44±11.39	92.65±12.67

*P<0.05 significant. SEM: Standard error of mean, TBARS: Thiobarbituric acid-reactive substances, GPX: Glutathione peroxidase, CAT: Catalase

In Table 4, a comparison was made between the levels of oxidative stress (enzymatic and nonenzymatic) before and after therapy in the study group. The results showed a statistically significant decrease in the levels of oxidative stress after the treatment.

Table 4

Comparison of antioxidant levels before and after treatment within the case group

Case	Mean±SEM
	TBARS	GPX	CAT
Before treatment	9.98±1.42*	187.44±22.75*	151.64±20.54*
After treatment	5.93±1.16	128.23±19.23	114.16±13.45

*P<0.05 significant. SEM: Standard error of mean, TBARS: Thiobarbituric acid-reactive substances, GPX: Glutathione peroxidase, CAT: Catalase

DISCUSSION

Oxidative stress-related tissue injury is the cause of various diseases in humans. Recently, many studies have been published using biomarkers to illustrate the pathogenesis of the disease.[1516] In the present study, marked difference was seen in the clinical parameters of the control and study groups [Table 1]. Baltacioglu et al.[17] conducted a study, in which he suggested that oxidative stress resulting from neutrophil hyperactivity has a vital role in severe periodontal destruction in AgP. Various ROS such as hydroxyl radical (OH-), hypochlorous acid (HOCl–), hydrogen peroxide (H2O2), and singlet oxygen (O2-) predominate in the extracellular environment, thus causing disease. These can be quantified using CAT, GPX as biomarkers. It also leads to the oxidation of lipids and DNA; proteins which can be assessed by quantifying TBARS are products of oxidative damage of lipids.[456] Table 2 shows marked improvement in the clinical parameters in the study group before and after mouth disinfection, which was comparable to a study done by Khorsand et al.[18]. Mechanical debridement and full mouth disinfection of the mouth lead to the removal of etiology, thus decreasing the oxidative stresses in the periodontal tissue. Direct relationships have been established between PI and probing depth by various studies.[19] As the inflammation subsides, there is probing depth reduces, and it leads to attachment gain.

Table 3 shows that there was a marked increase in TBARS, GPX and CAT levels in the study group before disinfection. TBARS is a signal of lipid peroxidation. Lipid peroxidation by free radicals results in the alteration of structural integrity and function of cell membranes.[20] A study by Panjamurthy et al.[21] showed increased plasma levels of TBARS in blood plasma gingival crevicular fluid (GCF) and gingival tissue locally in patients with periodontitis. Several other studies[2223] have shown the association between increased TBARS levels and deteriorating periodontal status in the saliva of adults.

GPX and CAT are the major ROS cleaning enzymes and have a significant role in protecting the periodontal tissues against oxidative stress. They inhibit the cytotoxic oxidative damage, protein degeneration, and lipid peroxidation.[24] Levels of GPX in the study group were higher than the control group, which was as per the study by Wei et al.[25] They reported high levels of the total amount of GPX in GCF samples from patients with gingivitis and periodontitis compared to healthy participants.

Table 4 depicts that the levels of plasma GPX decrease markedly after initial therapy; these results were similar to the study conducted by Patel et al.[26] that showed raised GPx levels in plasma and GCF in periodontitis as compared to the healthy periodontium. They suggested that it could be due to increased ROS generation at the diseased site. Similar results were given by Borges et al.[27] and Arunachalam et al.[6] Panjamurthy et al.[21] and Arunachalam et al.[6] in their studies showed the higher plasma levels of CAT in patients with periodontitis, which was similar to our results. Conversely, Gharbi et al.[28] showed a significant decrease in erythrocyte CAT activity. These changes can be related to the severity or spread of the disease. Arunachalam et al.[6] showed decreased in the levels of TBAR, CAT, and GPX after initial periodontal therapy, which was in accordance with our study.

The results of the present study showed that there is a direct relation between oxidative stress processes and the immune response, and disturbance in the balance leads to the decrease of AO, thus managing oxidative stress and improving periodontal health.

CONCLUSION

The results are in agreement with earlier studies showing that an increase in enzymatic and nonenzymatic oxidative stress may have a role in the pathogenesis AP and disinfecting the mouth or any other treatment can lead to the reduction of these stresses.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.