The biochemical analysis of saliva is becoming indispensable in dentistry. Monitoring salivary bio markers for oral and systemic diseases could become an important complement to clinical examination. Current clinical diagnostic parameters that were introduced more than a century ago continue to function as the basic model for Periodontal diagnosis. Biomarkers , whether produced by normal healthy individuals or by individuals affected by specific systemic diseases are tell tale molecules that could be used to monitor health status, disease onset , treatment response and outcome. This review limits itself to proteomic analysis of saliva in the diagnosis of periodontal diseases.
Clinical diagnosis of periodontal disease is based on oral examination consisting of inspection of the gingival tissue, conducting a periodontal screening and recording pocket depths for each tooth, checking attachment level, measuring plaque index, testing bleeding on probing, testing tooth mobility & taking radiographs to assess bone loss. All these are informative to evaluate disease severity but provide few useful determinants of disease activity.1,2,3 Periodontal disease severity may be ascertained by the salivary level of periodontal pathogens / host response markers and the periodontopathic bacteria may be acquired from the infectious saliva of close family members.4,5,6 The periodontopathic bacterial species identified in whole saliva includes Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Treponema denticola. Samples of both whole saliva & periodontal pockets may be needed to demonstrate/detect oral A.actinomycetamcomitans and T. forsythia as both of these can persist in non dental sites.7,8
Umeda et al compared the presence of six species of periodontopathic bacteria in whole saliva and sub gingival plaque from 202 subjects. The organisms were more frequently present in whole saliva than in periodontal pockets. The study found a relationship between the presence of periodontopathic bacteria in whole saliva and in periodontal pockets.9, 10
Saliva can be easily collected, contains locally derived and systemically derived markers of periodontal disease and hence may offer the basis for a patient specific diagnostic test for periodintitis. 11 12 Salivary analysis may offer a cost effective approach to assess periodontal disease incidence in large populations.13, 14
Recently there has been a growing interest in exploring the protein composition using advanced proteomics technology. The proteome is the protein complement of the genome and proteomics is analysis of the protein of the genome that is expressed. Human salivary proteomic analysis can provide information about the changes in the early stage of disease and monitor disease progression. Human salivary proteome analysis is important for understanding oral health and disease pathogenesis. Most relevant to periodontal disease are the emerging tool boxes of the salivary proteome and the salivary transcriptome for early detection, disease progression and therapeutic monitoring. Researchers have identified 1,1666 salivary proteins ,914 from the parotid fluid and 917 from the combined submandibular & sublingual fluids.15
The various proteomic bio markers used for periodontal disease monitoring and treatment are discussed below.
Immunoglobulins are important specific defence factors of saliva.Saliva contains secretory Ig A [S Ig A], IgG and Ig M.IgA2 is found in higher concentrations in tears , saliva and milk.16
Patients with periodontal disease are shown to have higher salivary concentrations of IgA, Ig G and IgM specific to periodontal pathogens compared with healthy patients.17-20 Saliva from treated periodontitis patients had higher IgA and IgG levels to Porphyromonas gingivalis and T denticola than as compared to saliva from controlled subjects.Increased concentrations of salivary IgG to A. actinomycetamcomitans in patients of aggressive periodontitis was reported by Sandholm et al .21
Salivary esterase has been statistically associated with calculus formation. Salivary esterase levels were found to be useful in determining the efficacy of periodontal treatment. Lysozyme is an anti microbial enzyme with the ability to cleave chemical bonds in the bacterial cell wall. It may also cause lysis of bacterial cells by interacting with monovalent anions and with proteases found in saliva. Patients with low levels of lysozyme in saliva are more susceptible to plaque accumulation, which is considered a risk factor for periodontal diseases.22 Peroxidase is a salivary enzyme produced by acinar cells in the salivary glands. This enzyme removes toxic hydrogen peroxide produced by oral micro organisms and reduces acid production in the dental biofilm. Patients with periodontal disease have demonstrated high levels of this enzyme in saliva.23Chitinase plays a role in the defence against chitin containing pathogens. Van steijn et al reported that chitinase level was raised in the saliva of periodontitis patients which decreased on treatment.24
Arginase is an arginine depleting enzyme.Significant variation in salivary arginase activity in periodontitis patients was reported by Ozmeric et al in 2000.25Beta glucuronidase in saliva is an indicator of neutrophil influx into gingival tissues and may provide as a risk factor in periodontal disease.26
A significant positive correlation between salivary acid phosphates & calculus formation was observed by Draus et al.27 It was found that mixed whole saliva of adult periodontitis patients revealed lowest enzyme activities. Other enzymes ,proteins and host response indicators in gingival crevicular fluid & saliva have been extensively explored using proteomic methods for their utility in periodontal disease diagnosis and include beta –galactosidase, beta and alpha glucosidase, caprylate esterelipa elastase, kininase, lactate dehydrogenase, myeloperoxidase, caprylate esterase, amino peptidase, aspartate aminotransferase, lactate dehydrogenase, cathepsin B , CD 14 , kallikrein.28-31 Nitric oxide ,which is a free radical with important cellular functions is produced and released from human neutrophils and macrophages. The increased salivary arginase activity causes a decrease in nitric oxide synthesis further leading to the decrease in the anti bacterial property of saliva, causing periodontal tissues to become more susceptible to existing pathogens.25
Calcium is the ion that has been most extensively studied as a periodontal marker for periodontal disease in saliva. A high concentration of calcium in saliva of periodontitis patients was observed by Sewon et al.32
Enzymes, proteins and immunoglobulin are the most abundant constituents of saliva. Recent studies have explored their value as biomarkers using proteomic methods.33,34,35 Mucins are glycoproteins produced by submandibular and sublingual glands and numerous minor salivary glands. The mucin MG2 affects bacterial aggregation and adherence and has a reverse relation with A. actinomycetamcomitans colonization.36 Lactoferrin is an iron binding glycoprotein produced by salivary glands .An elevated level of lactoferrin is seen in saliva of subjects affected by periodontitis compared with healthy subjects.37 Histatin is a salivary protein .It is an inhibitor of host and bacterial enzymes involved in the destruction of the periodontium. Histatin also inhibits the release of histamine from mast cells.38,39 Fibronectin is a glycoprotein that mediates inflammation, wound healing and tissue repair. Cystatins are proteolytic enzymes having collagenolytic activity. They may function by modulating enzyme activity in the periodontium.
Syrjanen et al reported that elevated levels of certain aminoacids especially proline may be detected in periodontitis patients. This may be the result of bacterial metabolism/degradation of salivary proteins rich in proline. In contrast the same authors also reported no diagnostic significance of aminoacids in periodontal diseases.39