Periodontitis, diabetes mellitus, and the lopsided redox balance: A unifying axis.

AIM: The aim of present study was to evaluate and compare the total antioxidant capacity in the saliva of type 2 diabetes mellitus (DM) patients and healthy subjects, with and without periodontal disease.
MATERIALS AND METHODS: The study was designed as a case-control study, comprising of 120 male subjects, who were divided into four groups of 30 patients each. Group I: Thirty type 2 diabetic males with periodontal disease; Group II: Thirty type 2 diabetic males without periodontal disease; Group III: Thirty healthy males with periodontal disease; Group IV: Thirty healthy males without periodontal disease. After clinical measurement and sampling, the total antioxidant capacities in the saliva of type 2 diabetic and healthy men were determined, and the data were tested by non-parametric tests. The total antioxidant capacity of the clinical samples was determined spectrophotometrically.
RESULTS: The total antioxidant capacity in the saliva was the lowest in type 2 diabetic males with periodontal disease. The results were statistically significant.
CONCLUSION: The findings of our study finally conclude that the salivary total antioxidant capacity is affected in type 2 diabetic males, in addition to the impact of periodontal disease, and hence, can be used as a useful marker of periodontitis in healthy and diabetic patients.

INTRODUCTION

Oxygen is required for all living organisms for their survival. However, at the same time it is potentially toxic. Salvemini has described oxygen as a double-edged sword. It is vital to life, but at the same time because of it's highly reactive nature it is capable of becoming a part of the potentially damaging molecules called free radicals. Oxygen is the ultimate electron acceptor in the mitochondrial electron transport chain, where the flow of electrons ultimately produces energy in the form of adenosine triphosphate. The leaked out electrons are exposed to oxygen leading to the formation of free radicals.[1]

A free radical has one or more unpaired electrons in its structure, which makes it extremely reactive toward other molecules.[2] The reactive oxygen species or free radicals can damage cells by a variety of mechanisms, including peroxidation of lipid membranes, protein inactivation, induction of DNA damage, and oxidation of important enzymes.[2]

The living organism has adapted itself to an existence under a continuous efflux of free radicals. Among the different adaptive mechanisms the antioxidant defense mechanism is of major importance.[3]

An antioxidant is any substance that when present at low concentrations, compared to those of an oxidizable substrate, significantly delays or prevents the oxidation of that substrate.[2] Living cells are exposed to antioxidants originating from a large variety of exogenous or endogenous sources.

The different possible mechanisms by which antioxidants may offer protection against free radical damage include:[4] Prevention of the formation of free radicals, interception of free radicals by scavenging the reactive metabolites and converting them into less reactive molecules, facilitating the repair of damage caused by free radicals, and providing a favorable environment for the effective functioning of other antioxidants.

Diabetes mellitus is a group of complex multisystem metabolic disorders characterized by a relative or absolute insufficiency of insulin secretion and or concomitant resistance to the metabolic action of insulin on target tissues.[5]

Hyperglycemia is a hallmark of diabetes mellitus, as are its chronic metabolic complications.[6] Hyperglycemia induces oxidative stress, which is an imbalance between the production of reactive oxygen species or free radicals and the antioxidant defense mechanisms present in biological systems. Oxidative stress is demonstrated by an increase in the reactive oxygen species levels and a drop in antioxidant defenses.[7]

Periodontitis, is a term used to describe an inflammatory process, the primary etiological agent is specific, predominantly gram-negative, anaerobic or facultative bacteria within the subgingival biofilm. Bacteria can cause tissue destruction directly by toxic products and indirectly by activating host defense mechanisms.[8]

Among the host responses leukocytes serve as the initial host defense against periodontal pathogens. After stimulation by bacterial pathogens, the neutrophils produce free radicals. Damage mediated by free radicals can be mitigated by an ‘Antioxidant Defense System’. Physiological alteration and pathological states produced by free radicals depend on the disequilibrium between free radical production and antioxidant levels leading to oxidative stress. Periodontal tissue destruction is caused by an inappropriate host response to these microorganisms and their products. More specifically due to oxidative stress.[1]

Periodontal diseases are one of the most common oral diseases worldwide. They are the single principle cause of tooth mortality in human beings. Although now it has been unanimously accepted that periodontal disease is the resultant of an interaction between microbial plaque and the resultant inflammatory and immunological changes within the periodontal tissues, it is also recognized that the nature and severity of this interaction in turn may be modified by many systemic factors.[9101112]

Among the systemic factors, the relationship between periodontal disease and diabetes mellitus has been studied extensively. Many investigators, in their epidemiological, experimental, and clinical studies have reported that the prevalence and severity of periodontal diseases is significantly greater among diabetics than among non-diabetics.[1314]

lt has been recognized that saliva serves as a mirror of the body's health, as it contains proteins, hormones, antibodies, and other molecules that are frequently measured in the standard blood tests to monitor health and disease.[15] However, unlike whole blood, saliva is easy to collect, less painful to the patient, and is less infectious for the health care provider.[15]

We hypothesized that a diabetic state would reduce the salivary antioxidant capacity of the subjects, and furthermore, this antioxidant impairment may help to explain the association between diabetes and inflammatory periodontal disease.

The aim of this article was to estimate and compare the total antioxidant capacity in the saliva of type 2 diabetic men and healthy subjects with and without periodontal disease

Aims and objectives

This study was conducted with the following aims

To estimate the total antioxidant capacity of saliva in type 2 diabetic men with and without periodontitis

To estimate the total antioxidant capacity of saliva in healthy men with and without periodontitis

To compare the total antioxidant capacity of saliva in type 2 diabetic men and healthy subjects with and without periodontal disease.

MATERIALS AND METHODS

A total of 120 male subjects between the ages of 40 and 65 years, who reported to the Department of Periodontics, A.B. SHETTY MEMORIAL INSTITUTE OF DENTAL SCIENCES and from the diabetic clinic of K.S. HEGDE MEDICAL ACADEMY, Mangalore, were included in the study.

Sixty patients with type 2 diabetes mellitus and no other systemic disease that could affect periodontal status participated in this cross-sectional study. Of these, group I included 30 type 2 diabetic men with periodontal disease and group II included 30 type 2 diabetic men without periodontal disease.

The patients with diabetes were those consecutively referred during medical care visits from an outpatient diabetic clinic (K.S. Hegde Medical Academy). All the patients with diabetes were diagnosed as having type 2 diabetes using the American Diabetes Association diagnostic criteria.[16] These patients were not under any oral hypoglycemic agents and/or insulin therapy (Fresh cases).

In addition, 60 systemically healthy subjects of whom 30 healthy men with periodontal disease (group III) and 30 healthy men without periodontal disease (group IV – control group) were recruited from those patients seeking dental treatment at the Department of Periodontics, A.B. Shetty Memorial Institute of Dental Sciences.

Patients were excluded if they had aggressive periodontitis, <14 teeth present, history of antibiotic therapy within the preceding three months, periodontal treatment within the last six months, smokers, persons who were obese, on vitamin supplements, and under insulin therapy/oral hypoglycemic medication.[17]

This study was approved by our ethics committee. The study protocol was explained and informed written consent was received from each individual before his enrollment in the study.

Clinical procedure

Dental examination

Clinical periodontal parameters, including plaque index, probing depth, clinical attachment level, and bleeding on probing were assessed. All clinical examinations were carried out by a single examiner, who was trained, calibrated, and masked to the systemic condition of the patient. Each tooth was measured and examined for probing depth (PD) in millimeters and clinical attachment level (CAL) in millimeters at six sites per tooth (mesiobuccal, buccal, distobuccal, mesiolingual, lingual, distolingual) using a Williams graduated periodontal probe. The dental plaque was scored as being present or absent at four points (mesial, buccal, lingual and distal) on each tooth. Bleeding on probing (BOP) was assessed at the six sites, at which PD was determined and was deemed positive if it occurred within 15 seconds after probing. BOP was expressed as the percentage of sites showing bleeding. Periodontal health was defined as the absence of gingival pockets ≥ 4 mm and absence of attachment loss ≥ 3 mm, with no BOP. Periodontal disease was defined as two or more tooth sites with PD ≥ 4 mm or CAL of 4 mm that bled on probing.[18]

Saliva sampling

All saliva samples were obtained in the morning after an overnight fast. The subjects were requested not to drink [except water] or chew gum for the same period and abstention was checked prior to the biological sample collection.[19]

For the collection of saliva the subject was seated in the coachman's position, head slightly down and was asked not to swallow or move his tongue or lips during the period of collection. The saliva was allowed to accumulate in the mouth for two minutes and he or she was asked to spit the accumulated saliva into the receiving vessel.[20] Two milliliters of unstimulated saliva was collected and stored at a temperature of 4°C in plastic or glass vials. The collected saliva was subjected to analysis using a spectrophotometer.[21]

The total antioxidant capacity of the saliva was evaluated using the spectrophotometric assay.[21] The method is based on the principle that, when a standardized solution of the Fe-EDTA complex reacts with hydrogen peroxide by a Fenton-type reaction, it leads to the formation of hydroxyl radicals (OH). These reactive oxygen species degrade benzoate, resulting in the release of thiobarbituric acid reactive substances (TBARS). Antioxidants from the added sample of human fluid cause suppression of the production of TBARS. This reaction can be measured spectrophotometrically and the inhibition of color development is defined as the antioxidant capacity.[21]

Statistical analysis

All the data obtained were subjected to statistical evaluation using the Student's unpaired t test and one way ANOVA. A value of P < 0.01 was considered to be significant. All values are expressed as mean ± SD. For these procedures a statistical program SPSS 11.0 was used.

RESULTS

Table 1 displays the clinical periodontal variables in the study groups. As expected, the mean PD and CAL were higher in diabetic patients with periodontal disease (P < 0.001). They also exhibited a higher percentage of sites with PD ≥ 4 mm, sites with CAL ≥ 4 mm, sites with plaque, and sites exhibiting BOP (P < 0.01).

Table 1

Clinical and periodontal characteristics of the subjects (mean±SD range)

The total antioxidant levels were significantly lower in diabetic patients with periodontitis [mean of 0.4020 ± 0.9561] compared to diabetic patients without periodontitis (mean of 1.2457 ± 0.18049) (P < 0.01) [Table 2].

Table 2

Mean and standard deviation of the total antioxidant level in diabetic patients with and without periodontitis

Analysis of the total antioxidant capacity revealed higher antioxidant levels in healthy individuals without periodontitis (mean of 4.6807 ± 0.31605) when compared to individuals with periodontitis (mean of 1.0917 ± 0.19090) and the levels were found to be statistically highly significant (P < 0.01) [Table 3].

Table 3

Mean and standard deviation of the total antioxidant level in healthy individuals with and without periodontitis

The comparison of the total antioxidant capacity revealed lower antioxidant levels in diabetic patients with periodontitis (mean of 0.4020 ± 0.09561) compared to healthy individuals with periodontitis (mean of 1.0917 ± 0.1909) (P < 0.01) [Table 4].

Table 4

Mean and standard deviation of total antioxidant levels between diabetes and healthy individuals with periodontitis

The total antioxidant levels decreased significantly in diabetic patients (mean of 1.2457 ± 0.18049) compared to healthy controls (mean of 4.6807 ± 0.31605) (P < 0.01) [Table 5].

Table 5

Mean and standard deviation of total antioxidant levels between diabetes and healthy individuals without periodontitis

Comparing the total antioxidant levels among the four groups the lowest values were observed in diabetic patients with periodontal disease, whereas, the highest values were present in healthy subjects without periodontal disease.

The saliva flow rates were similar in both the groups of diabetic patients (Groups 1 and 2). Although there were no significant differences between periodontitis subjects and controls (Groups 3 and 4), the salivary flow rates were significantly less in diabetic patients than the systemically healthy group [Table 6].

Table 6

Salivary flow rates

DISCUSSION

For a clinician, saliva means ‘whole saliva’, which is the fluid present in the mouth and comprises of not only pure secretions from the major and minor salivary glands, but also gingival exudates, microorganisms, and their products, epithelial cells, food remnants, and also to some extent nasal exudates.[22] Saliva may constitute a first line of defense against free radical-mediated oxidative stress, since the process of mastication promotes a variety of such reactions including lipid peroxidation.[23]

Unstimulated saliva samples were used as they were preferred over stimulated saliva for determination of the antioxidant defense parameters, and moreover, it has been claimed that the total antioxidant capacity is higher in unstimulated saliva.[24]

Saliva possesses a wide range of antioxidant defense molecules, including uric acid, vitamin C (ascorbic acid), reduced glutathione (GSH), oxidized glutathione (GSSG), and others.[25] Such antioxidants work in concert rather than alone, and hence, the total antioxidant capacity may be the most relevant parameter for assessing the defense capabilities.[26] Investigations of individual antioxidant activities may be misleading and less representative of the whole antioxidant status. Moreover, the number of different antioxidants makes it difficult, and also expensive, to measure each of them separately.[27]

The current evidence indicates that periodontal disease occurs in predisposed individuals with an aberrant inflammatory/immune response to microbial plaque. Chronic inflammatory conditions are generally thought to be associated with increased oxidative stress, with phagocytes, particularly neutrophils, (the most prominent cells of the gingival inflammatory infiltrate in patients with periodontitis) being implicated in disease pathogenesis, because of the generation of an oxidative burst during phagocytosis and killing.[2]

Originally, ROS were thought to be directly microbicidal, but recent evidence indicates that their role is to establish an environment in the phagocytic vacuole suitable for killing and digestion by enzymes released into the vacuole from cytoplasmic granules.[28]

Locally within the periodontal tissues, the excessive release of ROS and alteration in redox balance can result in local tissue damage directly (e.g., oxidation of extracellular and cellular macromolecules) and indirectly via activation of the redox-sensitive nuclear transcriptions such as NFkB and AP-1, leading to an amplification of the inflammatory and immune processes.[2930]

Data on the salivary total antioxidant capacity are conflicting. Moore et al.[31] measured the antioxidant capacity of saliva in periodontally diseased and healthy individuals, but failed to find any significant differences in saliva between the groups. Sculley and Langley–Evans[32] reported that periodontal disease is associated with reduced salivary antioxidant status and increased oxidative damage within the oral cavity.

When we compared the total salivary antioxidant capacity with respect to flow rate, a significantly lower rate of antioxidant production was noted in patients with periodontal disease compared to the healthy matched controls, suggesting that these patients either had a lower threshold of antioxidant defense capacity and/or were predisposed to exaggerated ROS-release at the peripheral sites.

Diabetes mellitus type-2 is a multicausal disease, which develops slowly and in a stepwise order.[333435] Persistent hyperglycemia, auto-oxidation of glucose, obesity, glycation of proteins, activation of NADPH oxidase, nitric oxide synthase, and xanthine oxidase cumulatively contribute to a free radical pool in DM.[363738]

The excessive levels of ROS produced in diabetes are the proximal step in the activation of stress-sensitive signaling pathways (e.g., NFκB), with other cell-signaling pathways (hexosamine and PKC), which are also associated with the upregulation of pro-inflammatory cytokines,[39] diabetic complications,[40] and insulin resistance.[41]

Patients with diabetes commonly present with xerostomia[31] and we found lower salivary flow rates in patients with type 2 diabetes mellitus compared to systemically healthy controls.

We also observed that the mean total antioxidant capacity of saliva was significantly lower in diabetic patients compared to the healthy control group, indicating that chronic hyperglycemia resulted in the saturation of cellular antioxidant capacity, with the ongoing stimulation of redox-sensitive cell signaling pathways and downstream activation of biochemical pathways associated with the development of diabetes and diabetic complications.

Periodontitis has been identified as the sixth complication of diabetes[42] and its prevalence in type 2 diabetic patients is more than twice that of non-diabetic patients.[4344] Diabetic patients display an increased severity of disease.[4546] However, periodontitis appears to have a reciprocating negative impact on the diabetic status[4748] and significant relationships between both periodontitis and impaired glucose tolerance[49] have been reported.

The current evidence points to a bidirectional interrelationship between diabetes and periodontitis. An upregulated inflammatory state has been proposed as the common mechanism underlying both conditions, with an increase in cytokines, including TNF-α, postulated as a possible link.[5051]

The results of our study demonstrated lower levels of total antioxidant capacity in the saliva of diabetic patients with periodontitis when compared to diabetic patients without periodontitis.

We suggest that oxidative stress is a common factor in periodontal disease and type 2 diabetes, and that the imbalance in redox control resulting independently from these disease states acts synergistically and amplifies the biochemical and clinical course of these diseases in a bidirectional manner.

A larger cohort study by Sculley and Langley-Evans,[32] found a lower total antioxidant capacity in the stimulated saliva samples from women than men and also in periodontitis subjects versus controls. The gender differences were confirmed by Brock et al.[26] for saliva and also for serum and plasma total antioxidant capacity. In the Sculley and Langley-Evans study, women had significantly lower saliva flow rates than men, which may explain the gender-specific differences in the total antioxidant capacity. The urate levels were also lower in women than men, which given the large contribution of urate to saliva total antioxidant capacity,[31] may also contribute to the lower overall salivary total antioxidant capacity in females.

Hence, to reduce the potential confounding factors, a tight matching for gender, age, periodontal status, and exclusion of smokers was done in this study. To the best of our knowledge, this is the first reported investigation of possible differences in the total antioxidant capacity in periodontally healthy and diseased diabetic males, who were diagnosed for the first time [fresh cases] and were not under any medication.

The principal limitations of our study include the small sample size and the lack of serum and gingival crevicular fluid data on antioxidants. However, these were beyond the scope of the present study, and evaluation of the antioxidant profile in saliva was the particular goal. Evaluation of serum and/or gingival crevicular fluid samples could be the aim of a separate study, and compiling of a group of diabetics presenting with clinically healthy periodontium is also difficult, because most of the patients with diabetes have some degree of inflammatory periodontal disease.

CONCLUSION

Oxidative stress lies at the heart of periodontal tissue damage, which results from host microbial interactions, either

As a direct result of excessive reactive oxygen species activity

Antioxidant deficiency

Activation of transcription factors and the creation of a proinflammatory state

Even as a myriad of possible mechanisms leading to periodontal destruction exist, the influence of free radicals and antioxidants cannot be overlooked undoubtedly.

Several avenues of enquiry now exist for the development of antioxidant-based approaches to periodontal therapy, which include traditional routes of increasing the antioxidant capacity of periodontal tissues and newer routes based on the modulation of transcription factors.

The oxidative status of diabetic patients, both systemically and locally, within the periodontal tissues needs to be considered as part of the treatment regimes. Efforts to develop therapeutic strategies aimed at limiting ROS production or increasing the rate of removal by antioxidant mechanisms in diabetic patients have been advocated.

Patients with diabetes need to be informed of their increased risk for periodontitis. Periodontal therapy should be a key consideration in the management of diabetic patients with comorbid periodontitis, due to the potential negative impact of periodontitis on the local and systemic oxidative status and glycemic control. The importance of maintaining optimal glycemic control in an effort to minimize metabolically generated ROS with its consequent deleterious effects on the periodontal tissues, should also be emphasized to these patients.