|Year : 2013 | Volume
| Issue : 2 | Page : 214-218
Chromogranin A: Novel biomarker between periodontal disease and psychosocial stress
Arunima Padmakumar Reshma1, Rajeev Arunachalam1, Jayakumar Kochu Pillai2, Sarath Babu Kurra3, Vini K Varkey4, Mohanraj J Prince1
1 Department of Periodontics, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari, Tamil Nadu, India
2 Department of Biochemistry, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari, Tamil Nadu, India
3 Department of Pharmacology, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari, Tamil Nadu, India
4 Department of Prosthodontics, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari, Tamil Nadu, India
|Date of Submission||15-Mar-2012|
|Date of Acceptance||22-Mar-2013|
|Date of Web Publication||6-Jun-2013|
Arunima Padmakumar Reshma
Department of Periodontics Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
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.
Keywords: Biomarkers, chromogranin A, chronic periodontitis, neuroendocrine hormones, plasma, psychosocial stress, saliva
|How to cite this article:|
Reshma AP, Arunachalam R, Pillai JK, Kurra SB, Varkey VK, Prince MJ. Chromogranin A: Novel biomarker between periodontal disease and psychosocial stress. J Indian Soc Periodontol 2013;17:214-8
|How to cite this URL:|
Reshma AP, Arunachalam R, Pillai JK, Kurra SB, Varkey VK, Prince MJ. Chromogranin A: Novel biomarker between periodontal disease and psychosocial stress. J Indian Soc Periodontol [serial online] 2013 [cited 2021 Apr 12];17:214-8. Available from: https://www.jisponline.com/text.asp?2013/17/2/214/113076
| Introduction|| |
Periodontal diseases have multifaceted pathogenesis caused by specific periopathogenic microorganisms and their metabolic products.  They lead to destruction of the periodontal ligament and alveolar bone with periodontal pocket formation gingival recession or both.  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. , Several studies showed that the psychological stress and psycho-social factors are involved in the etiology of periodontal disease. ,, Stress has long been regarded as an important pre-disposing factor for acute necrotizing ulcerative gingivitis (ANUG).  This appears to be true for HIV - associated Severe ANUG as well.  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.  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.  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.  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.  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.  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.  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.  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.  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.  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.
- 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)  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.
Assessment of psychosocial stress status
- 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.
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.
In the morning following an overnight fast, saliva samples were collected from subjects. The method as described by Navazesh  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.
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.
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.
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  and Plaque accumulation were recorded by Silness and Loe 1964. 
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 [Figure 2] and [Figure 3]. However, there is no significant difference between CgA levels in saliva and plasma.
|Figure 1: Comparison of mean and standard error of mean vales of periodontal clinical parameters between case and control groups|
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|Figure 2: Comparison of mean plasma chromogranin A levels between case and control groups|
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|Figure 3: Comparison of mean salivary chromogranin A levels between case and control groups|
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|Table 1: Comparison of mean and standard error of mean vales of periodontal clinical parameters between case and control groups|
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|Table 2: Comparison of CgA levels in plasma and saliva between case and control groups|
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| 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.  Animal studies demonstrated a possible detrimental role of stress in periodontal tissue.  Gaspersic et al.  found less attachment and more alveolar bone loss after exposing the experimental animals to restraint stress. Breivik et al.  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.,  Hilgert et al.  ) and a case-control study Moss et al.  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 Th 2 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 ) 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.  Some important functions of CgA inside the neuroendocrine cells include regulation of granulogenesis and hormone storage.  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.  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|
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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. , 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.  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.  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.  Daniel et al.  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.  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  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.  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.
| Acknowledgments|| |
The authors thank, Department of Biochemistry, University of Kerala for their valuable help in biochemical analysis of our samples.
| References|| |
|1.||Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: Current concepts. J Periodontol 1992;63:322-31. |
|2.||Kornman KS, Page RC, Tonetti MS. The host response to the microbial challenge in periodontitis: Assembling the players. Periodontol 2000 1997;14:33-53. |
|3.||Genco RJ, Löe H. The role of systemic conditions and disorders in periodontal disease. Periodontol 2000 1993;2:98-116. |
|4.||Genco RJ. Current view of risk factors for periodontal diseases. J Periodontol 1996;67 Suppl (10) 1041-49. |
|5.||Moulton R, Ewen S, Thieman W. Emotional factors in periodontal disease. Oral Surg Oral Med Oral Pathol 1952;5:833-60. |
|6.||De Marco TJ. Periodontal emotional stress syndrome. J Periodontol 1976;47:67-68. |
|7.||Davis CH, Jenkins CD. Mental stress and oral disease. J Dent Res 1962;41:1045-49. |
|8.||Giddon DB, Zackin SJ, Goldhaber P. Acute necrotizing ulcerative gingivitis in college students. J Am Dent Assoc 1964;68:381-6. |
|9.||Winkler JR, Robertson PB. Periodontal disease associated with HIV infection. Oral Surg Oral Med Oral Pathol 1992;73:145-50. |
|10.||Biondi M, Zannino L G. P sychologicalstress, neuroimm-unomodulation, and susceptibility to infectious diseases in animals and man: A review. Psychother Psychosom 1997;66:3-26. |
|11.||Breivik T, Thrane OS. Psychoneuroimmune interactions in periodontal disease. Psychoneuroimmunology 2001;63:627-44. |
|12.||Brogden KA, Guthmiller JM, Salzet M, Zasloff M. The nervous system and innate immunity: The neuropeptide connection. Nat Immunol 2005;6:558-64. |
|13.||Hironaka M, Ansai T, Soh I, Ishisaka A, Awano S, Yoshida A, et al. Association between salivary levels of chromogranin A and periodontitis in older Japanese. Biomed Res 2008;29:125-30. |
|14.||O'Connor DT, Frigon RP. Chromogranin A, the major catecholamine storage vesicle soluble protein. Multiple size forms, subcellular storage, and regional distribution in chromaffin and nervous tissue elucidated by radioimmunoassay. J Biol Chem 1984;259:3237-47. |
|15.||Taupenot L, Harper KL, O'Connor DT. The chromogranin- secretogranin family. N Engl J Med 2003;348:1134-49. |
|16.||Simon JP, Aunis D. Biochemistry of the chromogranin A protein family. Biochem J 1989;262:1-13. |
|17.||Nobels FR, Kwekkeboom DJ, Coopmans W, Schoenmakers CH, Lindemans J, De Herder WW, et al. Chromogranin A as serum marker for neuroendocrine neoplasia: Comparison with neuron-specific enolase and the alpha-subunit of glycoprotein hormones. J Clin Endocrinol Metab 1997;82:2622-8. |
|18.||Soell M, Feki A, Hannig M, Sano H, Pinget M, Selimovic D. Chromogranin A detection in saliva of type 2 diabetes patients. Bosn J Basic Med Sci 2010;10:2-8. |
|19.||Holmes TH, Rahe RH. The Social Readjustment Rating Scale. J Psychosom Res 1967;11:213-8. |
|20.||Navazesh M, Kumar SK. Xerostomia: Prevalence, diagnosis, and management. Compend Contin Educ Dent 2009;30:326-328. |
|21.||Muhlemann HR, Son S. Gingival sulcus bleeding-a leading symptom in initial gingivitis. Helv odontol Acta 1971:15:107-113. |
|22.||Silness J, Loe H. Periodontal disease in pregnancy. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964:22:112-120. |
|23.||Shklar G, Glickman I. The effect of cold as a stressor ag-ent upon the periodontium of albino rats. Oral Surg Oral Med Oral Pathol 1959;12:1311-20. |
|24.||Gaspersic R, Stiblar-Martincic D, Skaleric U. Influence of restraint stress on ligature-induced periodontitis in rats. Eur J Oral Sci 2002;110:125-9. |
|25.||Breivik T, Sluyter F, Hof M, Cools A. Differential susceptibility to periodontitis in genetically selected Wistar rat lines that differ in their behavioral and endocrinological response to stressors. Behav Genet 2000;30:123-30. |
|26.||Hugoson A, Ljungquist B, Breivik T. The relationship of some negative events and psychological factors to periodontal disease in an adult Swedish population 50 to 80 years of age. J Clin Periodontol 2002;29:247-53. |
|27.||Hilgert JB, Hugo FN, Bandeira DR, Bozzetti MC. Stress, cortisol, and periodontitis in a population aged 50 years and over. J Dent Res 2006;85:324-8. |
|28.||Moss ME, Beck JD, Kaplan BH, Offenbacher S, Weintraub JA, Koch GG, et al. Exploratory case-control analysis of psychosocial factors and adult periodontitis. J Periodontol 1996;67:1060-9. |
|29.||Corti A, Gasparri A, Chen FX, Pelagi M, Brandazza A, Sidoli A, et al. Characterisation of circulating chromogranin A in human cancer patients. Br J Cancer 1996;73:924-32. |
|30.||Blaschko H, Comline RS, Schneider FH, Silver M, Smith AD. Secretion of a chromaffin granule protein, chromogranin, from the adrenal gland after splanchnic stimulation. Nature 1967;215:58-9. |
|31.||Giampaolo B, Angelica M, Antonio S. Chromogranin 'A' in normal subjects, essential hypertensives and adrenalectomized patients. Clin Endocrinol (Oxf) 2002;57:41-50. |
|32.||Itagaki K, Kannan KB, Livingston DH, Deitch EA, Fekete Z, Hauser CJ. Store-operated calcium entry in human neutrophils reflects multiple contributions from independently regulated pathways. J Immunol 2002;168:4063-9. |
|33.||Daniel MA, McDonald G, Offenbacher S, Van Dyke TE. Defective chemotaxis and calcium response in localized juvenile periodontitis neutrophils. J Periodontol 1993;64:617-21. |
|34.||Zhang D, Shooshtarizadeh P, Laventie BJ, Colin DA, Chich JF, Vidic J, et al. Two chromogranin a-derived peptides induce calcium entry in human neutrophils by calmodulin-regulated calcium independent phospholipase A2. J PLoS One 2009:4:4501-3 |
|35.||Parekh AB, Putney JW Jr. Store-operated calcium channels. Physiol Rev 2005;85:757-810. |
|36.||Ciesielski-Treska J, Ulrich G, Chasserot-Golaz S, Zwiller J, Revel MO, Aunis D, et al. Mechanisms underlying neuronal death induced by chromogranin A-activated microglia. J Biol Chem 2001;276:13113-20. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]