|Year : 2013 | Volume
| Issue : 1 | Page : 42-46
Salivary protein concentration, flow rate, buffer capacity and pH estimation: A comparative study among young and elderly subjects, both normal and with gingivitis and periodontitis
Mulki Shaila1, G Prakash Pai2, Pushparaj Shetty3
1 Department of Oral Pathology & Microbiology, K V G Dental College, Sullia, Karnataka, India
2 Department of Periodontics, K V G Dental College, Sullia, Karnataka, India
3 Department of Oral Pathology, A B Shetty Memorial Institute of Dental Sciences, Deralkatte, Mangalore, Karnataka, India
|Date of Submission||09-Mar-2011|
|Date of Acceptance||12-Sep-2012|
|Date of Web Publication||21-Feb-2013|
Department of Oral Pathology and Microbiology KVG Dental College and Hospital Kurunjibag, Sullia - 574 327 Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: To evaluate the salivary protein concentration in gingivitis and periodontitis patients and compare the parameters like salivary total protein, salivary albumin, salivary flow rate, pH, buffer capacity and flow rate in both young and elderly patients with simple methods. Materials and Methods: One hundred and twenty subjects were grouped based on their age as young and elderly. Each group was subgrouped (20 subjects) as controls, gingivitis and periodontitis. Unstimulated whole saliva was collected from patients and flow rate was noted down during collection of the sample. Salivary protein estimation was done using the Biuret method and salivary albumin was assessed using the Bromocresol green method. pH was estimated with a pHmeter and buffering capacity was analyzed with the titration method. Student's t-test, Fisher's test (ANOVA) and Tukey HSD (ANOVA) tests were used for statistical analysis. Results: A very highly significant rise in the salivary total protein and albumin concentration was noted in gingivitis and periodontitis subjects of both young and elderly. An overall decrease in salivary flow rate was observed among the elderly, and also the salivary flow rate of women was significantly lower than that of men. Conclusion: S ignificant associations between salivary total protein and albumin in gingivitis and periodontitis were found with simple biochemical tests. A decrease in salivary flow rate among elderly and among women was noted.
Keywords: Elderly, gingivitis, periodontitis, salivary protein
|How to cite this article:|
Shaila M, Pai G P, Shetty P. Salivary protein concentration, flow rate, buffer capacity and pH estimation: A comparative study among young and elderly subjects, both normal and with gingivitis and periodontitis. J Indian Soc Periodontol 2013;17:42-6
|How to cite this URL:|
Shaila M, Pai G P, Shetty P. Salivary protein concentration, flow rate, buffer capacity and pH estimation: A comparative study among young and elderly subjects, both normal and with gingivitis and periodontitis. J Indian Soc Periodontol [serial online] 2013 [cited 2020 Apr 5];17:42-6. Available from: http://www.jisponline.com/text.asp?2013/17/1/42/107473
| Introduction|| |
Saliva lacks the drama of blood, the sincerity of sweat and the emotional appeal of tears. Despite the absence of charisma, practitioners are finding that saliva provides an easily available, non-invasive diagnostic medium for a rapidly widening range of diseases and clinical situations. 
In the oral cavity, proteins, especially albumin, are considered as a serum ultrafiltrate to the mouth.  Salivary proteins have been shown to be increased in medically compromised patients whose general conditions get worse. Elderly subjects usually show less-effective immune response than the young ones. Gingivitis and periodontitis are oral diseases that are characterized by chronic inflammation. Here, salivary protein and albumin concentrations were determined as markers for plasma protein leakage, occurring as a consequence of the inflammatory process. Hence, the aim of the present study was to analyze the salivary total protein, albumin, pH, buffering capacity and flow rate in young and elderly subjects, both normal and with gingivitis and periodontitis, using simple biochemical methods.
| Materials and Methods|| |
Ethics committee approval and informed consent
The study was approved by the Ethics Committee of A. B. Shetty Memorial Institute of Dental Sciences. Written informed consent was obtained from each subject after the aims and methodology of the study were explained.
Subjects were chosen from the department of Oral Medicine and Radiology and Department of Periodontics, A.B. Shetty Memorial Institute of Dental Sciences. Eighty subjects were chosen on the basis of presence of gingivitis and periodontitis under two different age groups.
Criteria for gingivitis was based on the National Institute of Dental Research (NIDR) criteria
NIDR - Gingival Inflammation Index (Bleeding index)
1 = Bleeding after probe is placed in gingival sulcus up to 2 mm and drawn along the inner surface of the gingival sulcus.
Criteria for periodontitis was based on loss of attachment with pocket depth of >/=5 mm in at least eight sites.
The first group comprised of forty young subjects between twenty and thirty five years of age with no systemic diseases and not on medication. The second group comprised of forty elderly subjects of sixty five years and above, with no systemic diseases and not on medication. Twenty control samples for each group were collected on the basis of presence of healthy periodontium with no systemic diseases and not on medication.
All the patients were subjected to routine examinations and case history was recorded. Based on the above-mentioned criteria, subjects were subgrouped under six groups as:
- Young - Control (number of subjects = 20)
- Young - Gingivitis (number of subjects = 20)
- Young - Periodontitis (number of subjects = 20)
- Elderly - Control (number of subjects = 20)
- Elderly - Gingivitis (number of subjects = 20)
- Elderly - Periodontitis (number of subjects = 20).
Human whole unstimulated saliva was collected by spitting method without swallowing, with the subject seated in an upright position between 11 am and 12 noon, after they had refrained from oral intake, tooth brushing and smoking for 2 h before saliva collection. Approximately, 5 mL of saliva was collected. Flow rate was calculated as volume collected divided by the time required for the collection. Salivary protein estimation was done based on the Biuret method. Protein forms a colored complex with cupric ions in alkaline medium. Based on this principle, salivary protein estimation was done by mixing undiluted saliva with the reagent (45 g of Rochelle salt and 15 g of copper sulfate in 400 mL of 0.2 N sodium hydroxide. Five grams of potassium iodide was added to make up to 1 L with 0.2 N sodium hydroxide) and measuring the colored product using a photoelectric colorimeter at a wavelength of 546 nm. Standard solution of 6 g of bovine albumin dissolved in 100 mL of normal saline containing 0.1 g/dL sodium azide was used.
Salivary albumin was estimated using the Bromocresol green method (albumin colorimetric test). The reaction between albumin in saliva and the dye Bromocresol green (prepared by mixing 8.85 g succinic acid, 108 mg Bromcresol green, 100 mg sodium azide and 4.0 mL Brij-35 in 900 mL of distilled water) produces change in color, which is proportional to the albumin concentration in the saliva. It was estimated using a photoelectric colorimeter at wavelength of 630 nm. Standard solution of 6 gm of bovine albumin dissolved in 100 mL of normal saline containing 0.1 g/dL sodium azide was used.
Salivary pH was estimated electrometrically with the help of a pHmeter. A pair of electrodes (glass electrode and calomel electrode) was dipped in saliva, whereby potential developed across the thin glass of the bulb (of glass electrode). Variations of pH with electromotive force (E) were recorded directly on the potentiometer scale graduated to read pH directly.
Then, a titration method was used to determine the buffering capacity. One milliliters of saliva of known pH was taken in a test tube to which was added phenol red indicator. It was titrated against 0.1 N sodium hydroxide till the pH was raised by one unit. The color was compared with the standard buffer. Then, saliva was titrated against 0.1 N hydrochloric acid using methyl red indicator to lower the pH by one unit. The titer values were noted down. The buffering capacity of the saliva toward acidic and alkaline side was calculated. Buffering capacity = titer value × normality × 1000/1000 × 10.
| Results|| |
The biochemical values of this study were subjected to statistical analysis to specify the statistical differences between the groups and subgroups. Student's t-test, Fisher's test (ANOVA) and Tukey HSD (ANOVA) tests were used to compare and correlate different parameters in subgroups among the young and the elderly.
[Table 1] correlates the parameters of the subgroups among the young and elderly using t-test. Of all the parameters, flow rate in all the three subgroups was shown to be higher in the younger group (P=0.001, very highly significant) as shown in the above-mentioned graphs. Buffer capacity of the elderly controls was significantly (P=0.054) higher than that of the young control group. pH, salivary total protein and albumin estimations did not show any significant changes.
|Table 1: Correlation of the parameters in the subgroups among the young and elderly (t-test)|
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[Table 2] and [Figure 1] estimate the significance of different parameters in combined young and elderly groups using Fisher's test. Salivary total protein and albumin estimation were shown to be very highly significant (P=0.001) biochemical markers. Salivary total protein in the controls, gingivitis and periodontitis subgroup was 0.86 g/mL (SD=0.21), 1.19 g/mL (SD=0.23) and 1.59 g/mL (SD=0.48). Total mean salivary albumin for controls, gingivitis and periodontitis patients was 0.09 (SD=0.04), 0.24 (SD=0.09) and 0.44 (SD=0.12) mg/mL. pH and buffer capacity did not show any significant changes among the control, gingivitis and periodontitis groups. There was no significant difference in all the parameters except flow rate, which was found to be higher in males (P=0.001, very highly significant) than in females [Figure 2].
| Discussion|| |
There was a rise in the total salivary protein concentration in the gingivitis and periodontitis subgroup in both the groups. In total, the mean values in the controls, gingivitis and periodontitis subgroups were 0.86 g/mL (SD=0.21), 1.19 g/mL (SD=0.23) and 1.59 g/mL (SD=0.48). The rise in these values was statistically very highly significant (P=0.001). The study conducted by Henskens, et al. showed the mean values in the controls, gingivitis and periodontitis subgroup as 1.06 mg/mL (SD=0.25), 1.49 mg/mL (SD=0.58) and 2.21 mg/mL (SD=1.0). Both the groups showed 1.8 and 1.3 times value rise in the periodontitis and gingivitis subgroups, respectively, when compared with that of the controls.
In general, the major factors affecting the protein concentration and composition of whole saliva are the salivary flow rate, protein contributions of the glandular saliva and crevicular fluid proteins. Thus, the elevated protein levels are most likely due to enhanced synthesis and secretion by the individual glandular saliva. Also, glandular-derived proteins, Cystatin C and amylase showed significant rise in periodontitis subjects, proving the glandular origin of these proteins.  In addition, the rise in salivary albumin also plays a role in the rise of total proteins. Thus, in the present study, salivary total protein concentration was proved to be a valuable biochemical marker of periodontal disease using the Biuret method.
Albumin was formerly detected as a minor component of whole, parotid, submandibular and sublingual saliva. Notable rise in albumin concentration in the gingivitis and periodontitis subgroups was noted when compared with the controls using the Bromocresol green method. The groups showed around four to five and two to three times rise in the periodontitis and gingivitis subgroups, respectively, when compared with that of the controls. Total mean salivary albumin for controls, gingivitis and periodontitis patients was 0.09 (SD=0.04), 0.24 (SD=0.09) and 0.44 (SD=0.12) mg/mL. The rise in these values was statistically very highly significant (P=0.001). There are reports of studies in which increased albumin concentrations during inflammation and periodontal breakdown were found in saliva and gingival crevicular fluid (GCF). , It showed the mean values in the controls, gingivitis and periodontitis subgroups as 0.08 mg/mL (SD=0.05), 0.30 mg/mL (SD=0.30) and 0.67 mg/mL (SD=0.50), which are slightly on the higher side than that found in the present study. Comparison of the data between the groups showed that there was no significant difference. It signifies the fact that old age as such does not affect the composition of saliva. Saliva from totally edentulous patients contained five to six times less albumin than saliva from the controls, confirming the sulcular origin for albumin.  This suggests the role of this parameter as a marker for gingivitis and periodontitis where plasma protein leakage occurs as a consequence of the inflammatory process.
GCF is both a physiological fluid as well as an inflammatory exudate, originating from the gingival plexus of blood vessels in the gingival corium, subjacent to the epithelium lining of the dentogingival space. As GCF traverses through inflamed periodontal tissues en route to the sulcus, biological molecular markers are gathered from the surrounding areas and are subsequently eluted into whole saliva. 
The hypothesis that periodontal microbes trigger inflammatory response and results in higher levels of salivary albumin and total protein is well known. Therefore, proteins are considered potential markers for plasma protein leakage. Subjects with periodontitis had significantly more P. gingivalis, P. intermedia and T. denticola when compared with the controls. T. denticola increased the levels of salivary albumin and total protein as the proteins existing in the periodontal pocket, including immunoglobulins and albumin, are potential energy sources for T. denticola.  Hollman and Van Der Hoeven (1999) have reported that degradation of albumin by T. denticola strains was not detected, but suggested that T. denticola occurs in close association with strong proteolytic bacteria such as P. gingivalis and T. forsythia in the subgingival plaque.  Thus, controlling the microbes in turn decreases the inflammatory response, which in turn decreases the plasma leakage in the saliva through GCF.
Salivary values have been published for 629 patients from the University of Lund, Sweden, where the majority of the subjects had resting flow rates of 0.1-0.5 mL/min.  It is in accordance with the present study, where the mean flow rate in the young subjects was around 0.5 mL/min. Flow rate did not alter with the periodontal status of the subjects in both the groups. Salivary flow rates in the elderly were lower than in adults in general. It seems that old age as such does not cause diminished salivary flow. , It has also been suggested that there are some age-related alterations in salivary function. 
As shown in the present study, there was a significantly decreased flow rate in females when compared with males (P=0.001). This difference has been suggested to be due to the size of the salivary glands.  Also, in menopause, many women seem to suffer from xerostomia, which then ameliorates in older age. 
The salivary flow is directly related to the salivary consistency. Thus, greater the salivary flow, greater the consistency and greater the cleaning and diluting capacities; therefore, if changes in health cause a reduction in salivary flow, there would be a drastic alteration in the level of oral cleaning.  The normal salivary pH is from 6 to 7, and varies in accordance with the salivary flow from 5.3 to 7.8. There are various sources of hydrogen ions in oral fluids: secretion by the salivary glands in the form of organic and inorganic acids, production by the oral microbiota or acquisition through foods. These ions influence the equilibrium of calcium phosphates in the enamel. The higher the concentration of hydrogen ions, the lower the pH, and vice versa. 
An interesting finding was significant rise in the buffering capacity among the elderly controls when compared with the young. Galgut  in his study had tried to correlate between pH and gingivitis and periodontal pockets. Statistically significant correlations between gingivitis and pH did not exist, but significant correlations did exist between pH and periodontal pockets. Our finding correlates with Sewon, et al. studies, which suggests that pH and buffer capacity changes in gingivitis and periodontitis are not as consistent as GCF changes. This may be due to the dilution of the GCF contents in total saliva.
The development of new separation techniques and different mass spectrometry instrumental devices, as well as the great availability of specific reactants, offers ample choice to scientists for carrying out high-throughput proteomic studies and being competitive in the field today. Koss, et al. analyzed total saliva for its physical and chemical properties. The aim of their study was to identify salivary parameters that could identify different stages of the periodontal disease. Dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used for protein detection and zymography for type IV collagenase identification. Salivary flow rate, pH and buffer capacity showed similar values in all groups. Proteins were augmented in severe periodontitis, as also shown by SDS-PAGE. Hydroxyproline rose significantly in all periodontal groups as secretory immunoglobulin A significantly diminished compared with the control group. An increase in peroxidase was detected in moderate and severe periodontitis. All salivary samples contained 200-116-92 kDa gelatinases; minor bands at 66-31 kDa were also present in all periodontitis groups. Calcium levels showed significant differences between all periodontitis groups compared with the control group. Thus, they claimed that quantitative changes in the chemical composition of the saliva of patients with periodontal disease could be of significance in the diagnosis and progression of periodontal disease. 
Quantitative proteomics (two-dimensional SDS-PAGE) was used to investigate whole saliva from individuals with severe periodontitis and their proteomic profiles before and after periodontal treatment were compared. Results highlighted the predominant involvement of S100 proteins in the host response during periodontitis, identifying host defence components that have not been linked previously to this disease and suggesting new potential biomarkers for monitoring disease activity in periodontitis. 
| Conclusion|| |
A very highly significant rise in the salivary total protein and albumin concentration using the Biuret and Bromocresol green methods suggests the role of these simple methods in assessing these parameters as markers for gingivitis and periodontitis, where plasma protein leakage occurs as a consequence of the inflammatory process . However, a longitudinal study would be required to draw definite conclusions and prove the role of saliva as a prognostic indicator.
| Acknowledgment|| |
The authors would like to acknowledge Dr. Sucheta Kumari, Professor, Department of Biochemistry, for her guidance in the Biochemistry Laboratory.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]