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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 1  |  Page : 18-24

Salivary total protein levels among healthy controls, chronic gingivitis patients and chronic periodontitis patients


1 Department of Periodontology, University of Dental Medicine, Mandalay, Myanmar
2 Department of Periodontology, University of Dental Medicine, Yangon, Myanmar

Date of Submission23-Sep-2020
Date of Decision20-Nov-2020
Date of Acceptance07-Dec-2020
Date of Web Publication15-Feb-2021

Correspondence Address:
Kyaw Thiha
Professor & Head.Department of Periodontology, University of Dental Medicine, Mandalay
Myanmar
Thaw Htet Zin
Demonstrator, Department of Periodontology, University of Dental Medicine, Mandalay
Myanmar
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jorr.jorr_46_20

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  Abstract 


Background: Periodontal diseases are one of the most common chronic infectious and inflammatory diseases in the world that can be diagnosed by clinical, radiographic signs and some biomarkers. Saliva contains locally and systemically derived markers that can aid in the diagnosis of periodontal diseases. The aim of the present study was designed to evaluate salivary total protein levels in patients with chronic gingivitis and chronic periodontitis.
Materials and Methods: Five milliliters of unstimulated whole saliva samples was collected from a total of 113 individuals; each of the healthy and chronic gingivitis groups was included 39 participants and 35 patients in the chronic periodontitis group. Salivary total protein levels, gingival index (GI), papillary bleeding index (PBI), probing pocket depth (PPD), and clinical attachment level were recorded in the respective groups. Salivary protein estimation was done by direct ultraviolet absorption method, and determination was based on biuret method.
Results: The mean salivary total protein levels in the healthy, chronic gingivitis, and chronic periodontitis groups were 1.52, 2.58, and 6.30 g/dl, respectively. The salivary total protein levels of gingivitis and periodontitis patients were 1.6 and 4.2 times higher than healthy controls, which were statistically significant (P < 0.001). In addition, GI (r = 0.476) and PBI (r = 0.490) were significantly correlated with protein levels in gingivitis patients. However, PPD was significantly correlated with protein contents in only periodontitis patients (r = 0.387).
Conclusions: Increased total protein levels are related to the severity of periodontal diseases, and it may serve as a biomarker in inflammation of the periodontium.

Keywords: Gingivitis, healthy, periodontitis, saliva, salivary total proteina


How to cite this article:
Zin TH, Soe O, Thet YM, Tun S, Hein YM, Thiha K. Salivary total protein levels among healthy controls, chronic gingivitis patients and chronic periodontitis patients. J Oral Res Rev 2021;13:18-24

How to cite this URL:
Zin TH, Soe O, Thet YM, Tun S, Hein YM, Thiha K. Salivary total protein levels among healthy controls, chronic gingivitis patients and chronic periodontitis patients. J Oral Res Rev [serial online] 2021 [cited 2021 Apr 22];13:18-24. Available from: https://www.jorr.org/text.asp?2021/13/1/18/309441




  Introduction Top


Periodontal diseases are plaque-induced chronic inflammatory conditions which may lead to irreversible destruction of tooth-supporting tissue including alveolar bone if left untreated, and it is also affected about 15% of the adult population worldwide.[1] The goal standard for diagnostic measurement in periodontal disease can be provided useful information for current periodontal state, location, type, and severity. Recently used clinically diagnostic procedures include probing pocket depth (PPD), clinical attachment loss, and radiographic assessment of alveolar bone loss. These procedures can only be described as the past history of periodontal disease and cannot reveal current or future disease activity.[2]

In medicine, many clinical samples of systemic diseases can be screened by human body fluids such as blood, cerebrospinal fluid, saliva, sera, urine, vaginal secretion, sputum, peritoneal fluid, pleural fluid, and pericardial fluids that have been approved for definitive diagnosis and maintenance of disease state.[3]

Saliva is one of the body fluids and is secreted by major and minor salivary glands. Saliva has many complexes of proteins with different biological roles in digestion, host defense, and lubrication which are predominantly comprising proline-rich proteins, mucin, amylase, immunoglobulins, cystatin, statherin, and many antibacterial factors.[4] Human salivary proteins can have wide ranges of properties for immune response, taste perception, digestion, inhibition of proteinase, oral defense mechanisms, chemotaxis, and cellular homeostasis.[5]

In periodontal diseases, the marginal and sulcular gingivae act as a front-line area where this interaction between bacteria and host immune response takes place by aggregation, adherence, cell-killing, and inhibition of microbial metabolism. Such interactions might be maintained in oral ecological condition. It may be hypothesized that quantitative variation in salivary protein concentrations will affect oral disease prevalence.[6] In the oral cavity, albumin can be considered as a serum ultrafiltration to the mouth, and thus, salivary protein concentrations can be determined as markers for plasma protein leakage, occurring as a consequence of the inflammatory process.[7]

Kejriwal et al.[8] described that total protein levels in chronic gingivitis and chronic periodontitis patients were higher than healthy controls, which was statistically significant. Based on the Henskens et al. study,[9] the group with periodontal disease showed a 1.8 times increase in total protein content compared with the healthy group.

In conclusion, Wakde et al.[10] suggested that chronic gingivitis and chronic periodontitis patients increased in the levels of total protein using simple biochemical tests. Hence, this cross-sectional clinical study in quantitative estimation of salivary total protein levels may assess to find the reliability of total protein as a diagnostic marker in gingivitis and periodontitis and which will help in the determination of disease activity, prevention, and treatment for periodontal diseases.


  Materials and Methods Top


This study was approved by the Ethical and Research Committee of the University of Dental Medicine, Mandalay, and written informed consent was obtained from each individual, which was conducted in the Department of Periodontology, University of Dental Medicine, Mandalay. One hundred thirteen individuals were included in the study and divided into three groups: 39 patients with chronic gingivitis, 35 patients with chronic periodontitis, and 39 healthy controls. The groups were divided based on the condition of the periodontal tissues, which was evaluated by clinical periodontal examination including assessment of papillary bleeding index (PBI), gingival index (GI), PPD, and clinical attachment level (CAL).

Inclusion criteria

  1. Individuals with healthy periodontium
  2. Chronic gingivitis patients
  3. Chronic periodontitis patients who have at least 6 teeth with PPD >3 mm
  4. Individuals having a minimum of 20 natural teeth.


Exclusion criteria

Individuals who have:

  1. Presence of medically compromised conditions
  2. Any diseases affecting the salivary production (e.g., Down's syndrome and Sjogren's syndrome)
  3. Apparent oral infection and oral ulceration (e.g., herpes and Candida)
  4. Intake of drugs that can affect the salivary gland (e.g., hyoscine, pilocarpine HCL, antihistamine, and anticough)
  5. Smoking habits, betel quid chewing habit, and taking alcohol
  6. Pregnancy or lactation period
  7. Intake of antibiotics or anti-inflammatory drugs in the last 2 weeks
  8. Periodontal therapy other than standard prophylaxis during the previous 3 months.


Collection of saliva

Participants were instructed not to make toothbrushing or eating or drinking within 2 h before the saliva collection procedure. Human whole unstimulated saliva was collected by spitting method without swallowing while sitting in an upright position. Approximately 5 ml of saliva was collected and stored at 4°C until the laboratory procedures were resumed. And then, the saliva sample was centrifuged at 6000 rpm for 15 min [Figure 1]. Salivary total protein estimation was used by ultraviolet absorption method, and determination was done by biuret method. Protein reacts with cupric ions as colored complex in alkaline medium. Based on this principle, salivary total protein was determined by mixing undiluted saliva with the biuret reagent [Figure 2] and [Figure 3] and measuring the colored changes using biowave spectrophotometer [Figure 4] and [Figure 5] at a wavelength of 546 nm.
Figure 1: Centrifuge machine

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Figure 2: Biuret reagent and bovine serum albumin

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Figure 3: Samples with biuret reagent

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Figure 4: Biowave spectrophotometer

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Figure 5: Sample result with printing paper

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  Results Top


Pearson's correlation coefficient was used for correlation between clinical periodontal parameters and salivary total protein levels. The mean total protein levels in the healthy, chronic gingivitis, and chronic periodontitis groups were 1.52 (standard deviation [SD] = 0.77) g/dl, 2.58 (SD = 1.12) g/dl, and 6.30 (SD = 1.93) g/dl, respectively. [Figure 6] When the comparison of salivary total protein levels in the healthy, chronic gingivitis, and chronic periodontitis groups was tested by ANOVA, the data obtained by F-statistics (df) were 128.716 (2, 110) and P < 0.001 which is highly significant. [Table 1] Thus, the salivary total protein levels of chronic gingivitis patients were higher than normal controls but lower than chronic periodontitis patients. Moreover, GI (r = 0.476, P = 0.002) and PBI (r = 0.490, P = 0.002) were statistically significant and positively correlated with the salivary protein levels in gingivitis patients [Table 2][Figure 7] and [Figure 8]. In chronic periodontitis patients, GI (r = 0.275, P = 0.111), PBI (r = -0.026, P = 0.884), PPD (r = 0.387, P = 0.022), and CAL (r = 0.220, P = 0.204), respectively. Among these, a statistically significant positive correlation was found between total protein outputs with PPD.[Table 3][Figure 9].
Table 1: Comparison of salivary total protein levels among healthy controls, patients with chronic gingivitis, and patients with chronic periodontitis (n=113)

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Figure 6: Salivary total protein levels of the participants

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Table 2: Correlation between clinical periodontal parameters and salivary total protein levels in patients with chronic gingivitis (n=39)

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Figure 7: Correlation between gingival index and salivary total protein levels in patients with chronic gingivitis (n = 39)

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Figure 8: Correlation between papillary bleeding index and salivary total protein levels in patients with chronic gingivitis

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Table 3: Correlation between clinical periodontal parameters and salivary total protein levels in patients with chronic periodontitis (n=35)

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Figure 9: Correlation between probing pocket depth and salivary total protein levels in patients with chronic periodontitis (n = 35)

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  Discussion Top


Saliva gets mixed with locally and systemically derived markers of periodontal disease which can be easily and rapidly collected and does not require any specialized equipment or techniques. Besides, salivary inorganic and secretary immunological substances play an important role in protecting the oral cavity from infection?.[9]

The present study also determined salivary total protein levels in patients with chronic gingivitis and chronic periodontitis with using simple biochemical methods. The elevated protein levels of periodontal diseases are most likely due to enhanced synthesis and secretion by the individual glandular saliva. It has known protective actions against infection through immunoglobulins. Besides, certain salivary enzymes such as β-glucuronidase, aspartate aminotransferase, and alkaline phosphatase have been reported in increased concentrations in periodontal disease and can be regarded as contributors to initiation and progression of periodontal disease.[11]

In this study, the mean total protein level in the healthy group was 1.52 (SD = 0.77) g/dl, the chronic gingivitis group was 2.58 (SD = 1.12) g/dl, and the chronic periodontitis group was 6.30 (SD = 1.93) g/dl.

The results of the present study also agreed with Shaila et al.,[7] which showed that the mean values in the healthy, gingivitis, and periodontitis subgroups were 0.86 g/ml (86 g/dl), 1.19 g/ml (119 g/dl), and 1.59 g/ml (159 g/dl), respectively, and increased significantly (P = 0.001) in periodontal diseases. Besides, the present findings are harmonious with Wakde et al.,[10] which found that the salivary total protein levels in the healthy, gingivitis, and periodontitis groups were 0.72 g/ml (SD = 0.23), 1.05 g/ml (SD = 0.19), and 1.16 g/ml (SD = 0.23), respectively. Henskens et al.[9] investigated protein, albumin, and cystatin concentrations in saliva of healthy controls and patients with gingivitis or periodontitis, and they found that all the periodontally healthy controls had salivary protein concentration between 0.5 and 1.5 mg/ml (0.05 and 0.15 g/dl), with a mean value of 1.06 ± 0.25 mg/ml (0.106 ± 0.025 g/dl). On the contrary, gingivitis and periodontitis showed a mean value of 1.49 ± 0.58 mg/ml (0.146 ± 0.058 g/dl) and 2.21 ± 1.00 mg/ml (0.221 ± 0.1 g/dl), respectively. The salivary proteins were significantly higher in a diseased state than in healthy controls (P < 0.01).

The previous findings on salivary total levels were quite different, and this may be influenced by some factors such as protein detection methods, utilized reagents, races, genetic variations in salivary glands, and so on.

The present findings were also similar to the result of previous research by Kejriwal et al.[8] It stated that the total salivary protein contents of the gingivitis group had a mean of 3.82 mg/ml (0.382 g/dl) and the healthy group was 2.97 mg/ml (0.297 g/dl). The results showed that the total salivary protein contents of the gingivitis group were more increased than the healthy group.[8]

The present data were in agreement with the findings of Priadarsini et al.[12] and Karthiga et al.[13] Priadarsini et al.[12] found that the mean salivary total protein level in the healthy group was 2.26 g/dl, and the mean total protein level among the chronic periodontitis group was 10.69 g/dl, which was highly significant.

Furthermore, Karthiga et al.[13] described that the mean salivary total protein values in the control and periodontitis groups are 0.87 g/ml (SD = 0.21) (87 g/dl [SD = 21]) and 1.67 g/ml (SD = 0.48) (167 g/dl [SD = 48]). The rise in these values was statistically significant (P =0.001).

Recent findings found that chronic gingivitis and chronic periodontitis patients were 1.6 and 4.2 times higher in the total protein contents than healthy controls. Henskens et al.[9] suggested that both the gingivitis and periodontitis subgroups showed a 1.3 and 1.8 times rise in the total protein value, respectively, when compared with that of the healthy group. The increased levels could partly be also due to an increased serum ultrafiltration of plasma protein into saliva and the response of individual salivary gland for soft-tissue inflammation by promoting the synthesis and secretion of certain acinar proteins for enhancing the oral defense mechanism.

The pairwise comparison of salivary total protein levels among healthy controls and patients with chronic gingivitis and chronic periodontitis showed that the data were highly and statistically significant in all intragroup comparisons in the present study (P <0.001).

A total of 39 subjects with chronic gingivitis patients were measured salivary total protein levels with their clinical parameters in the current study. GI (r = 0.476, P = 0.002) and PBI (r = 0.490, P = 0.002) were statistically significant and positively correlated with the salivary protein levels in gingivitis patients. However, there was a weak statistically significant relationship between PPD and salivary protein output in chronic gingivitis (r = 0.206, P = 0.209).

GI and PBI could be considered as represented clinical parameters for the severity and extension of gingival inflammation.[14] Thus, the increased protein levels of gingivitis patients which correlated GI and PBI in the present study could be related to the inflammatory process that was activated the sympathetic system and enhanced the synthesis and secretion by individual glandular saliva. This process was also suggested by Henskens et al.[15]

Most of the gingivitis patients had normal PPD although salivary protein levels were increased in the present study. It can be assumed that the output of total protein weakly correlated with PPD in chronic gingivitis groups. The rises of protein level in gingivitis patients might be due to serum ultrafiltration of blood-derived proteins through the gingival sulcus into the whole saliva. This process was also agreed with Gonçalves et al.[16]

Gonçalves et al.[16] suggested that gingival inflammation was associated with increased amounts of blood-derived proteins (serum albumin and hemoglobin), immunoglobulin peptides, keratins, and higher prevalence of alpha-amylase fragments in the gingivitis group. And then, salivary cystatins were appeared to be more abundant in the healthy group than the gingivitis group.

There was some correlation between salivary protein outputs with clinical periodontal parameters of chronic periodontitis patients in the present study and had shown as GI (r = 0.275, P = 0.111), PBI (r = -0.026, P = 0.884), PPD (r = 0.387, P = 0.022), and CAL (r = 0.220, P = 0.204), respectively. Among these, a statistically significant positive correlation was found between total protein outputs with PPD in Pearson's correlation analysis.

The result of the present study was in accordance with the finding of Sánchez et al.[17] and they postulated that significant correlation for protein concentration with PPD (P<0.05) as r=0.13.

Although the severity of periodontal disease was determined by CAL, there might be a weak positive correlation between protein levels and CAL in the present data. It could be explained that some periodontitis patients had severe attachment loss with shallow pocket depth in a recent study and bacterial load would be low in those pockets. It might affect to interfere saliva–bacterium interaction, and this phenomenon would decrease the salivary protein output because salivary protein components played an important role in bacterial adhesion, agglutination, and substrate for bacterial growth. oral anaerobes microbes have some protease enzymes that attack salivary components such as collagen, fibronectin, and fibrinogen and may cause degradation of these protein for nutrition of oral microbes..[6] This protein–microbe interaction could take place within the deep periodontal pocket. Scannapieco, (1994) supported that output of salivary protein is strongly correlated with probing pocket depth (PPD) and weak correlation in clinical attachment level (CAL).[6]

Based on the research of the present study, salivary protein levels significantly increased in periodontal disease conditions. These component changes might contribute to understanding the biological properties of saliva in the pathobiology of oral diseases.


  Conclusion Top


The present finding suggests that the salivary total protein levels are considerably associated with some clinical parameters of gingivitis and PPD in periodontitis. Salivary protein levels can also reflect gingival inflammation, periodontal pocket formation, and severity of periodontal conditions.

Longer studies should be conducted in relationship of identified marker to nature of periodontal disease. Although the saliva-based diagnostic method has been still challenged, these will be promising as a future application in diagnostic for periodontal diseases and to prognosticate periodontal treatment outcomes.

Acknowledgment

I wish to extend my grateful acknowledgment to the Implementation and Clinical Research Committee, Department of Medical Research, Lower Myanmar, Ministry of Health and Sports, for financial support of this research.

Financial support and sponsorship

This study was financially supported by the Implementation and Clinical Research Committee, Department of Medical Research, Lower Myanmar, Ministry of Health and Sports, for financial support of this research.

Conflicts of interest

There are no conflicts of interest



 
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Eke PI, Thornton-Evans G, Dye B, Genco R. Advances in surveillance of periodontitis: The centers for disease control and prevention periodontal disease surveillance project. J Periodontol 2012;83:1337-42.  Back to cited text no. 1
    
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Persson GR, Page RC. Diagnostic characteristics of crevicular fluid aspartate aminotransferase (AST) levels associated with periodontal disease activity. J Clin Periodontol 1992;19:43-8.  Back to cited text no. 2
    
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Khurshid Z, Naseem M, Sheikh Z, Najeeb S, Shahab S, Zafar M. Oral antimicrobial peptides: Types and role in the oral cavity. Saudi Pharm J 2016;24:515-24.  Back to cited text no. 3
    
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Humphrey RT, Williamson SP. A review of saliva: Normal composition, flow and function. J Prosthetic Dentist 2001;85:162-9.  Back to cited text no. 4
    
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Mahadevan K, Velavan S. Analysis of salivary proteins as the biochemical indicators of nutritional status and salivary gland function. Int J Pharma Bio Sci 2013;4:689-94.  Back to cited text no. 5
    
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Scannapieco F. Saliva-bacterium interactions in oral microbial ecology. Critical Rev Oral Biol Med 1994;5:203-48.  Back to cited text no. 6
    
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Shaila MG, Pai P, Shetty P. Salivary protein concentration, flow rate, buffer capacity and estimation: A comparative study among young and elderly subjects, both normal and with gingivitis and periodontitis. J Indian Soc Periodontol 2013;17:42-6.  Back to cited text no. 7
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Kejriwal S, Bhandary R, Thomas B, Kumari S. Estimation of levels of salivary mucin, amylase and total protein in gingivitis and chronic periodontitis patients. J Clin Diag Res 2014;8:56-60.  Back to cited text no. 8
    
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Henskens YM, van der Velden U, Veerman EC, Nieuw Amerongen AV. Protein, albumin and cystatin concentrations in saliva of healthy subjects and of patients with gingivitis or periodontitis. J Period Res 1993;28:43-8.  Back to cited text no. 9
    
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Wakde YN, Singh A, Singh AV. Comparative evaluation of salivary flow rate, pH, buffering capacity total protein and albumin levels in chronic periodontitis patients: A clinico-biochemical study. Int J Health Sci Res 2018;8:62-6.  Back to cited text no. 10
    
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Newman MG, Takei HH, Klokkevold PR, Carranza FA. Newman and Carranza's Clinical Periodontology. 13th ed. China: Elsevier; 2019.  Back to cited text no. 11
    
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Priadarsini T, Gayathri R, Vishnu Priya V. Evaluation of salivary total proteins, albumin, globulin and A/G ratio among healthy individuals and patients with chronic periodontitis. Drug Invention Today 2018;10:1175-8.  Back to cited text no. 12
    
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Karthiga DG, Geetha RV, Vishnu Priya V, Gayathri R. Comparative analysis of salivary protein in individuals with and without periodontitis. Int J Pharm Sci Rev Res 2017;43:23-4.  Back to cited text no. 13
    
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Azizi A, Lawaf S. The assessment of periodontal parameters, salivary total protein and albumin contents in patients taking warfarin. Dent J Shiraz Univ 2010;10:32-5.  Back to cited text no. 14
    
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Henskens YM, van der Velden U, Veerman EC, van den Keijbus PA, van der Weijden GA, Timmerman MF, et al. Protein composition of whole and parotid saliva in healthy and periodontitis subjects. J Period Res 1996;31:57-65.  Back to cited text no. 15
    
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Gonçalves L, Da R, Soares MR, Nogueira FC, Garcia CH, Camisasca DR, et al. Analysis of the salivary proteome in gingivitis patients. J Period Res 2011;46:599-606.  Back to cited text no. 16
    
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Sánchez GA, Miozza VA, Delgado A, Busch L. Relationship between salivary mucin or amylase and the periodontal status. Oral Dis 2013;19:585-91  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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