Chromogranin A: Novel biomarker between periodontal disease and psychosocial stress.

CONTEXT: The psychosocial stress has long been regarded as a significant pre-disposing factor for periodontal disease. The association between the periodontal disease and the neuroendocrine hormones has been observed. Chromogranin A (CgA) is supposed to link the activity of the neuroendocrine system to local and systemic immune functions and to be related to periodontitis. AIMS: The aim of this study was to determine the CgA levels in saliva and plasma in periodontal health and disease and to assess their potential relationship to periodontitis. SETTINGS AND DESIGNS: In this case-control study, the association between periodontal disease and stress marker has been assessed.
MATERIALS AND METHODS: SIXTY SUBJECTS WERE CHOSEN FOR THIS STUDY: With case group comprising of 30 subjects with chronic periodontitis and control group comprising of 30 healthy subjects. Salivary and plasma CgA levels were determined by ELISA technique. Clinical parameters included were plaque index, papillary bleeding index and clinical attachment loss and probing depth. Correlation analysis was calculated by independent sample t-test.
RESULTS: Significantly higher CgA levels were found in saliva and plasma of patients with chronic periodontitis compared with healthy individuals (P < 0.05). No significant difference were observed between salivary and plasma CgA levels.
CONCLUSIONS: The elevated level CgA in the plasma and saliva of subjects with stress induced chronic periodontitis has yielded insights into biological plausible association between the psychosocial stress and chronic periodontitis. Thus, our results suggest that CgA is a useful biomarker for evaluating at least in part the etiopathogenesis of periodontitis.

INTRODUCTION

Periodontal diseases have multifaceted pathogenesis caused by specific periopathogenic microorganisms and their metabolic products.[1] They lead to destruction of the periodontal ligament and alveolar bone with periodontal pocket formation gingival recession or both.[2] The onset, progress, and severity of these pathomorphological changes are determined by the individual host response, the latter being susceptible to modulation by various factors. Epidemiological studies indicate a so-called risk factors and risk indicators that may be closely related to the emergence and progression of periodontitis.[34] Several studies showed that the psychological stress and psycho-social factors are involved in the etiology of periodontal disease.[567] Stress has long been regarded as an important pre-disposing factor for acute necrotizing ulcerative gingivitis (ANUG).[8] This appears to be true for HIV – associated Severe ANUG as well.[9] The biologic plausibility for such an association is supported by studies showing that psychosocial conditions may affect the host immune response, making the individual more susceptible to infections.[10] Psychosocial stress can down regulate the cellular immune response mainly by the activation of hypothalamo pituitary axis system.

It has been shown that emotional or psychological stress may influence immune activities directly via neuroendocrine messenger substances.[11] The presence of neuropeptides has been implicated as neurogenic promoter in various inflammatory processes modulating the activity of the immune system and the release of cytokines.[12] A recently studied stress – related hormone Chromogranin A (CgA) is reported to have a close relationship between the extent and severity of periodontitis as compared to healthy subjects in Saliva.[13] CgA is an acidic glycoprotein belonging to a family of regulated secretory proteins stored in the dense core granules of the adrenal medulla and of many neuroendocrine cells and neurons.[14] This protein was originally identified as the major soluble protein of the secretory granules of chromoffin cells, co-released by exo-cytosis with the catecholamines from the adrenal medulla and sympathetic nerve endings.[15] It has been proposed that CgA is a precursor of several biologically active peptides with pivotal roles in the regulation of the endocrine, metabolic and immune system.[16] Because of their ubiquitous distributions in neuroendocrine and nervous system tissues and the co-secretion with resident peptide hormones and biogenic amines, chromogranins are valuable indicators of sympathoadrenal activity and clinically useful markers of secretion from normal and neoplastic neuroendocrine cells.[17] A recent cross-sectional study have reported that CgA is over expressed in saliva of type 2 diabetic patients. This study demonstrated that psychological state of a patient has an impact on the production of specific proteins there by mirrors the pathogenic status of an individual.[18] To our knowledge limited information is available to explain the biological plausible association of the novel biomarker CgA in the pathogenesis of periodontal disease.

The purpose of this study is to investigate the levels of CgA in saliva and plasma of subjects with and without chronic periodontitis and to assess the influence of psychosocial stress on periodontal disease.

MATERIALS AND METHODS

A total of 60 subjects participated in this case-control study. The study was conducted from September 2011 to November 2011. Thirty subjects with clinical evidence of chronic periodontitis (cases) and thirty periodontally healthy subjects (control) were recruited from a private dental clinic Trivandrum Kerala. The study was approved by Institutional Ethics Committee, Kerala University, India. The study protocol was explained, and written informed consent was obtained from each individual before clinical periodontal examinations and sampling were carried out. Subjects who fulfilled the below mentioned inclusion criteria were recruited for both saliva and plasma sampling.

Inclusion criteria

The group of patients with chronic periodontitis had at least four teeth in each jaw with a probing depth of ≥5 mm

Number of sites involved should have ≥2 mm clinical attachment loss

Stress rating scale (Holmes and Rahe-1967)[19] should score more than 150+

The healthy control group had at least 20 teeth, probing depth of ≤3 mm and clinical attachment level of ≤1 mm in all the teeth examined.

Exclusion criteria

History of smoking in the past 6 months (self-reported)

Subject with known systemic diseases such as diabetes mellitus, rheumatoid arthritis, and renal diseases

Pregnant and lactating women

History of periodontal therapy within the past 6 months.

The use of antibiotics, steroids, non-steroidal anti-inflammatory agents in the past 6 months.

Assessment of psychosocial stress status

In our study Psychosocial stress levels were assessed based on Holmes and Rahe stress scale, which comprises of series of self-assessed questionnaires termed as “Life Change Units” that apply to events in the past year of an individual's life. Individual units were added and the final score will give a rough estimate of how stress affects health.

The score interpretations are as follows:

Score of 300+: At risk of illness

Score of 150-299: Risk of illness is moderate (reduced by 30% from the above risk)

Score <150: Only have slight risks of illness.

Saliva sampling

In the morning following an overnight fast, saliva samples were collected from subjects. The method as described by Navazesh[20] was used for saliva sampling; whole saliva samples were obtained by expectorating into polypropylene tubes. The salivary samples were centrifuged at 3000 rpm for 15 min and supernatant obtained were immediately frozen at 40°C and stored until required for further biochemical analysis.

Plasma sampling

The venous blood samples were collected in Ethylene diamene tetra acetic acid EDTA (1 mg/ml) coated test-tube using a standard venipuncture method. The plasma was separated from blood by centrifugation at 3000 rpm for 10 min. The plasma samples were then stored at 40°C until required for biochemical analysis.

Biochemical assay

The saliva and plasma CgA Assay was carried out by ELISA technique (ELISA Kit from Biovendor-Laboratory. USA). The Assay characteristics revealed: Limit of detection: 10 ng/ml, intra-assay precision: Correlation Value CV%:8.9, inter-assay precision: CV%:9.5. Reference interval based on normal distribution (95% double sided) and established by lab was: 43.14-121.23 ng/ml. Reference interval based on normal distribution (95%-double sided) and established by our lab was: 0.13-16.00 pmol/ml. All plasma and saliva sample were assayed in duplicate and results were expressed as mean ± SEM.

Clinical measurements

Subsequent to saliva sampling, clinical periodontal recordings included plaque index, and presence of bleeding on probing. Probing depth, clinical attachment levels were performed at six sites (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual) on each tooth present except for the third molars, using a William's periodontal probe (Hu-Friedy. Chicago, IL, USA). The clinical attachment level was assessed from the cement-enamel junction to the base of the probaeble pocket. Bleeding was assessed by papillary bleeding index by Muhlemann 1977[21] and Plaque accumulation were recorded by Silness and Loe 1964.[22]

Statistical methods

The data were analyzed by In the SPSS System (SPSS Inc. V 17.0 Chicago IL, USA). P < 0.05 was considered as statistically significant. The correlation analysis as calculated by independent sample t-test.

RESULTS

The clinical parameters of the case-control groups are shown in Table 1, Figure 1. The mean values for case and control groups are plaque index (2.41 ± 0.01 and 0.28 ± 0.01), papillary bleeding index (55.62 ± 0.72 and 6.27 ± 0.48), probing depth (6.76 ± 0.14and 1.23 ± 0.07), clinical attachment level (CAL) (6.93 ± 0.13 and 1.23 ± 0.07) respectively. The mean values were significantly higher in chronic periodontitis group than in the control group. The novel biomarker CgA levels between cases and controls [Table 2] showed a statistically significant difference with mean values 82.63 ± 0.2 ng/ml and 59.56 ± 0.24 ng/ml in plasma and (4.62 ± 0.13 pmol/ml and 0.12 ± 0.23 pmol/ml) in Saliva with P < 0.05 [Figures 2 and 3]. However, there is no significant difference between CgA levels in saliva and plasma.

Table 1

Comparison of mean and standard error of mean vales of periodontal clinical parameters between case and control groups

Figure 1

Comparison of mean and standard error of mean vales of periodontal clinical parameters between case and control groups

Table 2

Comparison of CgA levels in plasma and saliva between case and control groups

Figure 2

Comparison of mean plasma chromogranin A levels between case and control groups

Figure 3

Comparison of mean salivary chromogranin A levels between case and control groups

DISCUSSION

The association between periodontitis and stress related hormones has been largely overlooked in dental research. Stress has been linked to periodontal disease since the middle of the last century and most reports are of necrotizing forms of periodontal disease.[8] Animal studies demonstrated a possible detrimental role of stress in periodontal tissue.[23] Gaspersic et al.[24] found less attachment and more alveolar bone loss after exposing the experimental animals to restraint stress. Breivik et al.[25] demonstrated that periodontal disease susceptibility and progression could be explained, at least in part by brain-neuroendocrine – immune regulatory mechanisms. Genetically determined hypothalamus-pituitary- adrenal axis (HPA) reactivity seems to play a vital role, and a possible feedback is likely to occur from periodontal disease. Several cross-sectional studies (Hugson et al.,[26] Hilgert et al.[27]) and a case-control study Moss et al.[28] found that HPA Axis hyperactivation may increase the odds ratio for periodontal disease. During the activation of HPA Axis, the T-helper cells (Th) phenotype is influenced by inhibition of interleukin IL-12 and stimulation of IL-10 secretion by macrophages (the so-called Th2 dominant shift) will progress to periodontal disease. In the present case-control study, we found a significant association between periodontitis and stress status of an individual. The case group exhibit a moderate to high score (175-300+) in the social readjustment rating scale (Holmes and Richard H.Rahe[29]) as compared to the control group (score >150). Thus, the data obtained here may be interpreted such that there is a potential positive influence of psychosocial stress on periodontal disease. In our study, we demonstrated for the first time that CgA levels in plasma and saliva of patients with chronic periodontitis were significantly elevated as compared to healthy subjects.

CgA is a protein that belongs to a family of secretory peptides found in neuroendocrine cells and Immune cells. It is co-released exocytotically with many different neuroendocrine hormones and is regarded as a good marker of increased general neuroendocrine activity as elevated levels in the blood are linked to neuroendocrine activation and hyperplasia or neoplasia of neuroendocrine tissues.[29] Some important functions of CgA inside the neuroendocrine cells include regulation of granulogenesis and hormone storage.[15] In addition, recent studies have shown that CgA can function as a pro-hormone, giving rise to bioactive split products that may exert modulating effects in an autocrine, paracrine or endocrine manner.[30] The origin of elevated CgA levels in this study may be due to local secretion from the immune cells within the periodontium due to inflammatory or remodelling process [Figure 4].

Figure 4

Possible mechanism of action of chromograninA on periodontal disease

Saliva plays a major part in the maintenance of oral mucosal and dental health and changes in the amount and the quantity of saliva may alter the oral health status. Saliva is a fluid that can be easily collected, contains locally and systematically derived markers of periodontal disease. In this context, there are also some reports concerning CgA expression in saliva, stress factors and dry mouth syndrome.[1315] These studies show that the psychological state of a patient has an impact on the production of specific proteins and thereby mirrors a psychological state or pathological conditions.

CgA is a very stable molecule in blood samples and its immunoreactivity in plasma is not affected by repeated freezing and thawing or prolonged incubation at 37°C.[14] This stability makes CgA a reliable marker that is not vulnerable in handling the samples. However, there it has been reported there is significant variations in serum CgA during the day, with higher levels observed in the late afternoon and at night.[31] Most subjects in the current study had their blood sample taken before early afternoon, suggesting that the diurnal effects did not influence the results.

The paradigm shift in understanding the pathogenic mechanism of periodontitis has centered on the role of alterations in neutrophil functions. Calcium homeostasis and alterations in intracellular pH play a critical role in a variety of neutrophil functions including the chemotactic response and phagocytosis and selected intracellular signalling pathways.[32] Daniel et al.[33] have demonstrated that the release of intracellular sequestrated calcium in the early phase of calcium response appears to be intact in neutrophils in localized aggressive periodontitis and the second phase of calcium response, associated with membrane channel activation and influx of extracellular calcium appears to be compromised. Recently, a study by Zhang et al.[34] reported that CgA may play a role in calcium signalling serving as immunomodulators for an activation of polymorpho nuclear neutrophils PMN secretion.

Parekh and Putney[35] have reported that stress can stimulate PMN secretion, by the release of CgA and CgA derived peptides from the adrenal medulla. Stress stimulated PMN pathway resulted in oxidase activation, cell degranulation and priming response to a wide variety of pro-inflammatory molecules. Yet, another study by Ciesielki et al.[36] reported that CgA induces marked accumulation of nitric oxide and tumor necrosis factor TNF-a by microglial cells. Thus, it is speculated that CgA could be one of the natural factors that trigger the microglial immune response involved in neurodegeneration. Thus, from our study, it has to be emphasized that psychological state of patient has an impact on the production of specific proteins and thereby mirrors the influence of psychosocial stress on periodontal disease. Further, interventional studies will be required to explore the biological plausible association between the novel biomarker CgA and periodontal disease.

CONCLUSION

The elevated level CgA in the plasma and saliva of subjects with stress induced chronic periodontitis has yielded insights into biological plausible association between psychosocial stress and chronic periodontitis. Thus, our results suggest that CgA is a useful biomarker for evaluating at least in part the etiopathogenesis of periodontitis. Future studies should focus on the possible predictive value of CgA as a biomarker of prognosis in stress associated periodontitis.