Malondialdehyde as a Marker of Oxidative Stress in Periodontitis Patients.

BACKGROUND: Periodontology is a fast-evolving field with newer insights into traditional periodontal diagnosis. Advances in periodontal disease diagnostic research are moving toward methods whereby periodontal risk can be identified and quantified by objective measures such as biomarkers.
OBJECTIVES: The aim of this study was to evaluate malondialdehyde (MDA) levels in whole saliva of patients with chronic periodontitis.
MATERIALS AND METHODS: Whole saliva samples were collected from 85 patients: 30 patients with chronic periodontitis, 25 with gingivitis, and 30 periodontally healthy controls. To determine the clinical condition of each subject, the gingival index and clinical attachment level were measured. The salivary MDA levels were determined spectrophotometrically.
RESULTS: Higher salivary MDA levels (P < 0.005) were detected in patients with periodontitis compared to the healthy controls. There was no statistically significant difference in the salivary MDA levels between patients with gingivitis and healthy controls.
CONCLUSIONS: Higher salivary MDA levels seem to reflect increased oxygen radical activity during periodontal inflammation. Thus, MDA can be used as a marker of oxidative stress in patients with periodontitis.

INTRODUCTION

Periodontitis is an oral inflammatory disease that causes tissue destruction and bone loss. There is a paradigm shift in the concept of etiopathogenesis of periodontal disease. It is now believed that the pathogenic organisms in the plaque flora are responsible for disease initiation and inappropriate host response plays a vital role in disease progression. Thus, periodontitis is due to complex interactions between pathogenic bacteria and host immune response.[1]

The inflammatory and the immune responses to the bacteria and viruses that colonize the periodontal and associated tissues involve the systemic circulation and ultimately the peripheral systems of the body.[2] This creates a complex, bidirectional series of host–microbe interactions involving cellular and humoral factors and networks of cytokines, chemokines, and growth factors.[3] Although the primary etiologic agent is specific, predominantly gram-negative anaerobic or facultative bacteria within the subgingival biofilm, the majority of periodontal tissue destruction is caused by an inappropriate host response to the microorganisms and their products. More specifically, a lack of equilibrium between proteolytic enzyme, their inhibitors, reactive oxygen species (ROS), and the antioxidant defense systems causes oxidative stress. Oxidative stress is believed to cause cellular and molecular damage, which further leads to tissue destruction.[4]

ROS cause tissue damage by a variety of mechanisms, which include DNA damage, lipid peroxidation (through activation of cyclooxygenases and lipooxygenases), protein damage including gingival hyaluronic acid and proteoglycans, and oxidation of important enzymes (e.g., antiproteases, α-antitrypsin, stimulation of proinflammatory cytokine release by monocytes and macrophages by depleting intracellular thiol compounds and activating nuclear factor).

Oxidative stress generally appears after exposure to a relatively high concentration of ROS and/or a decrease in antioxidant defense system against ROS. It has been implicated as a major contributor to more than 100 disorders and more recently periodontitis.[56]

A major challenge in clinical periodontics is to find a reliable molecular marker of periodontal tissue destruction with high sensitivity, specificity, and utility.[3] The level of oxidative stress can be measured by assessing the production of malondialdehyde (MDA) levels. Earlier studies have demonstrated that it is used as marker in carcinoma.[7] Considering the previous facts, this study is designed as a single-centered, randomized controlled clinical trial to analyze the level of salivary MDA in periodontally healthy subjects and patients with gingivitis and chronic periodontitis.

MATERIALS AND METHODS

Source of data

The subjects to be studied were selected from patients seeking treatment at the Department of Periodontics and Oral Implantology, Sree Anjaneya Institute of Dental Sciences, Calicut, India. A total of 85 systemically healthy subjects aged between 18 and 45 years with untreated moderate-to-advanced gingivitis and periodontitis were selected. After ethical approval, all patients were verbally informed, and written informed consent was taken for participation in the study. They were divided into three groups as follows: Group I: 30 subjects with chronic periodontitis (at least four teeth with one or more sites exhibiting clinical attachment loss ≥4 mm, probing depth ≥4 mm, bleeding on probing); Group II: 25 subjects with gingivitis (bleeding on probing); and Group III: 30 periodontally healthy controls (no history of any periodontal disease).

Selection criteria

The inclusion criterion for the study was the presence of at least 20 teeth in systemically healthy subjects. The exclusion criteria were previous or current smokers, pregnant females, subjects who are on antioxidant supplements, subjects with history of antibiotics intake in the past 3 months, and subjects on anti-inflammatory medication.

At clinical examination, whole saliva sample was collected in a quiet room between 9 AM and noon, at least 2 h after food intake, and was obtained by expectorating into disposable tubes before clinical measurements. About 2 mL of whole saliva sample was collected in tubes and centrifuged immediately to remove cell debris (3000g for 10 min at 40°C). The supernatant was removed and analysis was done immediately.

Assay of MDA levels

MDA reagent was prepared by mixing 75 mg thiobarbituric acid, 15 g trichloroacetic acid, and 2.08 mL of 0.2 N HCl. The reaction mixture was warmed to dissolve the contents and stored at 4°C. To this sample, 1 mL TCA-TBA-HCl reagent was added.

The saliva sample was mixed with the MDA reagent. The obtained samples were then placed in a boiling water bath for 15 min. The solution was taken and the optical density of the pink color formed was read at 535 nm. The concentration of MDA in the sample was determined by plotting the obtained absorbance against the standard graph. The optical density of the pink color formed was directly proportional to the concentration of MDA in the given sample. The optical densities of the test samples were directly proportional to the concentration of MDA in the sample and were calculated by plotting against the standard graph and multiplied by the respective dilution factors; the final concentration was expressed as µM/100 mL.[8]

Statistical analyses

Differences in clinical parameters and age between healthy controls and chronic periodontitis group were analyzed by an unpaired t test. Differences in MDA levels between groups were analyzed by the Dunnett t test. A value of P < 0.005 was considered to be significant. All values are expressed as mean ± standard deviation.

RESULTS

Mean results of MDA levels in the whole saliva of periodontally healthy controls and subjects with gingivitis and periodontitis are shown in Table 1. Salivary MDA levels were significantly higher in chronic periodontitis group compared to the controls (P < 0.005) [Table 2].

Table 1

Comparison of the malondialdehyde levels between healthy subjects and those with gingivitis and periodontitis

Subjects	Mean	n	Standard deviation	P value
Healthy	89.45	30	46.47
Gingivitis	159.60	25	12.28	<0.001
Periodontitis	281.58	30	83.50	HS
Total	180.33	85	106.07404

HS = highly significant

Table 2

Multiple comparisons

Dunnett t (two-sided) test		Mean difference (I – J)	Significance	95% Confidence interval
(I) Subjects	(J) Subjects			Lower bound	Upper bound
		Gingivitis	Healthy	70.14100	0.089 (NS)	−9.6237	149.9057
Periodontitis	Healthy	192.12700*	< 0.001 (HS)	126.9994	257.2546

NS = non significant, HS = highly significant

*The mean difference is significant at the 0.05 level.

DISCUSSION

Several mechanisms of periodontal tissue destruction have been proposed and include a complex array of factors such as those derived from the immune response, direct bacterial influence, and the host system in response to this trauma. It has been suggested that as a result of stimulation by bacterial antigens, polymorphonuclear neutrophils produce and release a large amount of ROS, culminating in heightened oxidative damage to gingival tissue, periodontal ligament, and alveolar bone.[6] In recent years, the roles of ROS, lipid peroxidation products, and antioxidant systems have received a lot of attention in the pathology of periodontitis.

Proteins may be the target of attack by ROS, such as those derived from lipid peroxidation and/or by degradation products. The latter is the case of MDA or 4-hydroxynonenal that may form stable cross-linked products with specific amino acids.[8] Malondialdehyde or malonaldehyde (MDA) can be formed during lipid peroxidation of polyunsaturated fatty acids (PUFAs) by the action of human platelet thromboxane synthetase on prostaglandins PGH2, PGH3, and PGG2, and by the action of polyamine oxidase and amine oxidase on spermine. MDA is metabolized in the liver to malonic acid semialdehyde. This is unstable and spontaneously decomposes to acetaldehyde that is then converted to acetate by aldehyde dehydrogenase and finally to carbon dioxide and water.[910] After this, the next steps include changes in conformation, enzymatic activity, or binding as well as receptor inactivation, increased susceptibility to proteases, and changes in immunogenicity.[7]

Increased levels of MDA have previously been reported in inflamed periodontal tissue and may play a role in the destructive processes of periodontitis, thus implicating a role of ROS in periodontal pathogenesis.[10]

The balance between ROS and antioxidant mechanisms is likely to be important in periodontal pathogenesis, and imbalance can be caused by (1) increased ROS and inhibited antioxidant mechanisms and/or (2) decreased antioxidant capacity in diseased subjects. Recent medical and dental research in this area are geared toward the isolation of factors promoting free radical–mediated tissue injury in patients with periodontitis and its prevention by using specific nutrient antioxidants.[1112] This study focused on lipid peroxidation in periodontitis. It has evaluated the level of MDA in saliva samples in a cohort of patients with periodontitis and gingivitis, and compared to corresponding control groups. It showed that the level of MDA is significantly higher in patients with periodontitis compared to healthy subjects.

ROS has very short life span, so it is not easy to detect them. Nevertheless, ROS-related tissue destruction could be observed by the final product of lipid peroxidation, such as MDA. Gutteridge reported that the extent of tissue damage could be assessed by measuring the concentration of lipid peroxidation products and antioxidants. Local oxidative damage and antioxidant activity can be measured in GCF gingival tissue or saliva.[13]

MDA as a biomarker is the principal and most studied product of lipid peroxidation. In this study, it was observed that MDA levels in patients with periodontitis were significantly higher as compared with those of healthy subjects demonstrating that levels of oxidative stress are higher in pathological than in healthy conditions. This finding was consistent with other studies that indicted increased MDA level correlates with the presence of periodontal disease and acts as an oxidative stress marker in patients with periodontal disease.[1014] Thus, excessive local production of ROS leading to increased lipid peroxidation as measured by MDA may play a role in periodontal pathogenesis.

CONCLUSION

Within the limitations of this study, we can conclude that patients with periodontitis demonstrate more lipid peroxidation and a suppressed antioxidant response than patients with gingivitis and healthy controls. There was an increased production of MDA in disease than in healthy conditions. Thus MDA can be used as a marker to differentiate between health and disease, but due to small sample size and limitation of study period, we could not reach a cutoff value of MDA. And since other confounding factors such as age, underlying systemic illness, and any other chronic diseases that can lead to alterations in the pro-oxidant/antioxidant status were accounted for, it may be suggested that the increase in periodontal disease severity could tip this balance toward oxidative stress.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.