LIST OF ABBREVIATIONS

SALIVARY PROTEIN CONCENTRATION,
BUFFER CAPACITY AND pH ESTIMATION-
A COMPARATIVE STUDY AMONG YOUNG AND
ELDERLY SUBJECTS, BOTH NORMAL AND
WITH GINGIVITIS AND PERIODONTITIS
Dissertation Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Dental Surgery
BRANCH IV : ORAL PATHOLOGY AND MICROBIOLOGY
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA.
2003 – 2006 Dr. Shaila.M

SALIVARY PROTEIN CONCENTRATION,
BUFFER CAPACITY AND pH ESTIMATION-
A COMPARATIVE STUDY AMONG YOUNG
AND ELDERLY SUBJECTS, BOTH NORMAL
AND WITH GINGIVITIS AND PERIODONTITIS
By
Dr. Shaila.M
Dissertation Submitted to the Rajiv Gandhi University of Health
Sciences, Karnataka, Bangalore.
In partial fulfillment of the requirements for
the degree of
MASTER OF DENTAL SURGERY
in
ORAL PATHOLOGY AND MICROBIOLOGY
Under the guidance of
Prof. (Dr.) S.E.Shroff
Department of Oral Pathology and Microbiology
A. B. Shetty Memorial Institute of Dental Sciences
(A UNIT OF NITTE EDUCATION TRUST)
DERALAKATTE, MANGALORE-575018.
KARNATAKA - INDIA
2003-2006

Rajiv Gandhi University of Health Sciences, Karnataka
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation titled “SALIVARY
PROTEIN CONCENTRATION, BUFFER CAPACITY AND pH
ESTIMATION- A COMPARATIVE STUDY AMONG YOUNG
AND ELDERLY SUBJECTS, BOTH NORMAL AND WITH
GINGIVITIS AND PERIODONTITIS” is a bonafide and a genuine
research work carried by me under the guidance of
Prof.(Dr.)S.E.Shroff, H.O.D, Department of Oral Pathology and
Microbiology,A. B. Shetty Memorial Institute of Dental Sciences.
Date: Dr. Shaila.M
Place:Mangalore
ii

CERTIFICATE BY THE GUIDE
A. B. Shetty Memorial Institute of Dental Sciences,
DERALAKATTE, MANGALORE-575018,
KARNATAKA, INDIA.
Prof. (Dr.) S.E.Shroff M. D. S
Department of Oral Pathology
and Microbiology
CERTIFICATE
This is to certify that the dissertation “SALIVARY PROTEIN
CONCENTRATION, BUFFER CAPACITY AND pH
ESTIMATION- A COMPARATIVE STUDY AMONG YOUNG
AND ELDERLY SUBJECTS, BOTH NORMAL AND WITH
GINGIVITIS AND PERIODONTITIS” is a bonafide research work
done to my satisfaction by Dr.Shaila.M This work was carried out
during the period 2003-2006. This dissertation is submitted in partial
fulfillment for the award of the degree of Master of Dental Surgery in
Oral Pathology and Microbiology of Rajiv Gandhi University of
Health Sciences, Bangalore.
Date: Prof (Dr.) S.E.Shroff
Place:
iii

CERTIFICATE BY THE HEAD OF THE DEPARTMENT
&
ENDORSEMENT BY THE DEAN OF THE INSTITUTION
A. B. Shetty Memorial Institute of Dental Sciences,
DERALAKATTE, MANGALORE-575018,
KARNATAKA, INDIA.
CERTIFICATE
This is to certify that the dissertation “SALIVARY PROTEIN
CONCENTRATION, BUFFER CAPACITY AND pH
ESTIMATION- A COMPARATIVE STUDY AMONG YOUNG
AND ELDERLY SUBJECTS, BOTH NORMAL AND WITH
GINGIVITIS AND PERIODONTITIS” is a bonafide research work
done to my satisfaction by Dr.Shaila.M This work was carried out
during the period 2003-2006. This dissertation is submitted in partial
fulfillment for the award of the degree of Master of Dental Surgery in
Oral Pathology and Microbiology of Rajiv Gandhi University of
Health Sciences, Bangalore.
Prof. (Dr.) S.E.Shroff
Head of the Department of Oral
Pathology and Microbiology
Date:
Place: Mangalore
iv
Prof. (Dr.) N. Sridhar Shetty,
Dean/ Principal
Head of the Department
Prosthodontics.
Date:
Place: Mangalore

DECLARATION BY THE CANDIDATE
I hereby declare that the Rajiv Gandhi University of Health Sciences,
Karnataka shall have the rights to preserve, use and disseminate this
dissertation / thesis in print or electronic format for academic / research
purpose.
Date: Dr. Shaila.M
Place:Mangalore
© Rajiv Gandhi University of Health Sciences, Karnataka
v

ACKNOWLEDGEMENTS
I take this opportunity to express my deepest gratitude and sincere thanks to
my esteemed professor and guide, Prof. (Dr.) S.E. Shroff, Head of the Department
of Oral Pathology and Microbiology, A.B.Shetty Memorial Institute of Dental
Sciences, Deralkatte, for his guidance, encouragement, patience and help rendered
during this study as well as throughout my post graduate course.
I take great pride in expressing my heartfelt gratitude to the President of
NET, Sri. N.Vinaya.Hegde without whose encouragement and support I would not
have reached this position in my life.
It is with great honour and pride that I convey my honest gratitude to Prof.
(Dr.) N. Sridhar Shetty, our beloved Dean, A.B.Shetty Memorial Institute of Dental
Sciences, Deralkatte ,who has been a constant source of inspiration to me
personally since my graduation days, till today.
No words can express my gratitude to our beloved sir, Dr. Pushparaja
Shetty, Associate Professor; Department of Oral Pathology. He was always there
whenever I was in need of his academic guidance. His expertise and interest in the
subject has helped me to develop deep rooted interest in the subject.
I am greatly thankful to Dr. Lal P. Madathil, Associate Professor,
Department of Oral Pathology, for his valuable guidance throughout the course.
With his immense knowledge, he has always been a source of inspiration to me.
I am deeply grateful to my Co-guide, Prof. (Dr.) Nandini Manjunath, Head
of the Department, Department of Periodontics, A.J.Shetty Institute of Dental
Sciences for the keen interest that she has taken in my study and guiding me.
I am grateful to Dr.Sucheta.Rai for helping me wholeheartedly during my
study.If not for her guidance and affection, my study would have been impossible. I
also wish to express my sincere thanks to Dr.Rajendra Prasad, H.O.D, Department
of Oral surgery, Dr. Gopa Kumar, H.O.D, Department of Oral Medicine and
Radiology, and Dr Biju Tom, H.O.D, Department Of Periodontics for helping me in
my study.
vi

I am very much thankful to Dr. Kotian M.S, Assoc. Prof, KMC, Mangalore
for all the help rendered in the statistical analysis of my study.
My personal gratitude to all teaching staff- Dr. Ganapthy Bhat, Dr.
Mangesh, Dr. Prajwal, Dr. Nirmal and Dr. Simy for their help and constant
support.
I am very much thankful to the non-teaching staff Mrs. Sumathi, Mr.
Bhadra, Mrs. Geetha, Mrs. Jyothi, Miss. Roopa and Miss. Tulasi, Department of
Oral Pathology, A. B. Shetty Memorial Institute of Dental Sciences for their kind co-
operation.
I would like to thank my senior colleagues Dr. Srilatha and Dr.
Kumaraswamy Naik L. R, especially my batch mate Dr.Riaz Abbas Abdulla for
moral support and my dear junior colleagues, Dr. Asha , Dr.Vinod, Dr. Ajay and
Dr. Anitha for their affection and constant support.
I would like to thank my dear friend Dr Audrey for her patience, Dr.
Kumarswamy K. L, Dr M.Fiyaz and Mr. Rithesh for their timely help and
Dr.Roopa for her constant support.
I thank A1 printers, who executed printing and binding for my thesis. I am
most indebted to my patients who have co-operated during my study.
With guidance from my dear parents I take this opportunity to thank the
Almighty for his blessings he has showered on me since my first footstep till today
and surely forever.
Date:
Place: Mangalore Dr. Shaila.M
vii

LIST OF ABBREVIATIONS
ANOVA - Analysis of Variance.
Da - Daltons
e.m.f - Electro motive force
GCF - Gingival crevicular fluid
HSD - Honestly significance difference test
No. - Number
ns - Not significant
PMN - Polymorphonuclear leucocytes
PRP - Proline-rich proteins
S.D. - Standard Deviation
sig - Significant
t - Students ‘t’ test
vhs - Very highly significant
viii

Background and objectives
ABSTRACT
The objective of this study was to evaluate the salivary protein
concentration in gingivitis and periodontitis patients and comparing parameters like
salivary total protein, salivary albumin, pH, buffer capacity and flow rate in both
young and elderly patients, to assess the role of these parameters as diagnostic
markers.
Method
The study included 120 subjects, who were grouped based on their
age as Young and Elderly. Further subgroups of 20 subjects each were made as
Controls, Gingivitis and Periodontitis subjects under each group. Unstimulated
whole saliva was collected from patients of all the subgroups in both the groups.
Flow rate was noted down during collection of the sample. Parameters like salivary
total protein, salivary albumin, pH & buffer capacity were estimated to assess their
role as markers of periodontal disease. Salivary protein estimation was done using
Biuret method and salivary albumin was assessed using Bromocresol green method.
pHmeter was used to estimate pH & buffer capacity was estimated by titration .The
results were tabulated and analysed statistically .
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
ix

elderly. Flow rates, pH and buffering capacity did not alter with periodontal status.
An overall decrease in salivary flow rate was observed among the elderly and also
salivary flow rate of women was significantly lower than that of men.
Interpretation and conclusion
The present study suggests the role of salivary total protein and albumin
as markers for gingivitis and periodontitis where plasma protein leakage occurs as a
consequence of the inflammatory process. It can be assumed that old age as such
need not affect the composition of saliva, but a decrease in salivary flow rate among
elderly and among women is a common finding.
Keywords: saliva ;albumin; protein; pH; buffer capacity; flow rate; gingivitis;
periodontitis
x

TABLE OF CONTENTS
TITLE PAGE NO.
1. Introduction 1
2. Objectives 3
3. Review of Literature 4
4. Methodology 38
5. Results 44
6. Discussion 54
7. Conclusion 59
8. Summary 60
9. Bibliography 61
10. Annexures 76
xi

NO.
LIST OF TABLES
TABLE NO. PAGE
1. Estimated values of the parameters
Group: Young - Subgroup : Control
2. Estimated values of the parameters
Group: Young - Subgroup : Gingivitis
3 Estimated values of the parameters
Group: Young - Subgroup : Periodontitis
4 Estimated values of the parameters
Group: Elderly - Subgroup : Control
5 Estimated values of the parameters
Group: Elderly - Subgroup : Gingivitis
6 Estimated values of the parameters
Group: Elderly - Subgroup : Periodontitis
7 Comparison of parameters in subgroups of the young
using Fisher’s test.
8 Comparison of the parameters in different subgroups in the
young using Tukey HSD test
9. Comparison of parameters in subgroups of the elderly
using Fisher’s test
xii
88
89
90
91
92
93
44
45
46

10a Comparison of pH in different subgroups of elderly group
using Tukey HSD test.
.
10b Comparison of the parameters in different subgroups –
elderly using Tukey HSD test
11 Correlation of the parameters in the subgroups among the
young and elderly using t-test.
12 Estimating significance of the different parameters in the
study.
13 Comparing parameters among subgroups
14 Correlating parameters in subgroups among males and
females using t test.
15 Laboratory procedure for salivary total protein estimation.
16 Laboratory procedure for salivary albumin estimation.
xiii
47
47
48
49
50
50
80
83

NO.
LIST OF FIGURES
PHOTOGRAPHS PAGE
1. ARMAMENTARIUM USED IN SALIVARY
TOTAL PROTEIN ESTIMATION
2. COLORIMETER
3. ARMAMENTARIUM USED IN SALIVARY
ALBUMIN ESTIMATION
4. pHMETER
xiv
42
42
43
43

LIST OF GRAPHS
GRAPHS PAGE NO.
1. Comparison of the variables among subgroups.
2. Comparing variables among young and elderly
in Controls
3. Comparing variables among young and elderly
in Gingivitis patients
4. Comparing variables among young and elderly
in Periodontitis patients
5. Comparing pH among young and elderly in
different subgroups
6. Comparing buffering capacity among young
and elderly in different subgroups.
7. Comparing the gender variations among
variables
xv
51
51
52
52
53
53
53

1.INTRODUCTION
Saliva is not one of the popular body fluids. It lacks the drama of
blood, the sincerity of sweat and the emotional appeal of tears. Despite the absence
of charisma, a growing number of dental and medical practitioners are finding that
saliva provides an easily available, noninvasive diagnostic medium for a rapidly
widening range of diseases and clinical situations 1 .
Saliva is the principal defensive factor in the mouth, and a reduction in
its flow rate affects orodental health. A reduced salivary flow may cause a variety of
mostly unspecific symptoms and so the establishment of patient’s salivary flow is of
primary importance in dentistry 2 . Salivary hypofunction is associated with oral and
pharyngeal disorders and requires early diagnosis and intervention. It is important to
establish reference flow rates in various populations 3 .
In the oral cavity, proteins, especially albumin is considered as a serum
ultrafiltrate to the mouth 4. Salivary proteins have been shown to be increased in
medically compromised patients whose general conditions get worse.
Immunosuppression, radiotherapy and diabetes are examples of states in which high
concentrations of salivary albumin have been detected. It may be hypothesized that
salivary albumin can be used to assess the integrity of mucosal function in mouth.
1

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. Salivary albumin also increases prior to cancertherapy
induced stomatitis and can be used as a predictor of stomatitis 5 .
Hence aim of the present study was to analyse salivary total protein,
albumin, pH, buffering capacity and flow rate in young and elderly subjects, both
normal and with gingivitis and periodontitis.
2

2.OBJECTIVES
• To evaluate the salivary protein concentration in gingivitis and periodontitis.
• To compare the salivary total protein concentration, salivary albumin
concentration, pH, buffering capacity and flow rate in the young and elderly
patients both normal and with gingivitis and periodontitis.
• To assess the role of salivary proteins as a diagnostic aid in the detection of
loss of mucosal integrity.
3

3.REVIEW OF LITERATURE
Prior to the seventeenth century and the anatomic demonstrations by
Stenson and Wharton of the ducts that bear their name, salivary glands were thought
to be accessory excretory organs, that strained off the evil spirits of the brain. With
the realization that the glands could generate an external secretion, physicians who
practiced medicine (humoral pathology), based on the need to balance the body
humors (phlegm, blood, yellow bile and black bile)”bled, blistered, purged” and
stimulated salivation. This negative image of saliva however was not uniform. In the
cosmologies of ancient Egypt, Thoth the wise is said to have spat into the empty eye
socket of Horus, the Sun god to restore his vision. The New Testament tells us that
Jesus took the blindman by the hand and led him out of town to spit on his eyes and
restore his vision. Now it is recognized that saliva is a natural resource with many
functional capabilities that include food preparation, digestion, lubrication and
protection of the teeth and mucous membranes (Mandel, 1990) 1 .
The major salivary glands are the parotid glands, submandibular glands
and sublingual glands. The parotid glands have serous acinar cells and produce a
4

proteinaceous, watery secretion, secretion from sublingual gland is mucous, and
hence more viscous.Submandibular glands have both serous and mucous acinar cells
which produce saliva with lower protein content and higher viscosity than parotid
glands. Minor salivary glands are situated on the tongue, palate, buccal and labial
mucosa. They are small mucosal glands with primarily mucous secretion. The
working part of the salivary gland tissue consists of the secretory end pieces (acini)
and the branched ductal system. The fluid first passes through intercalated ducts
which have low cuboidal epithelium and narrow lumen. Then the secretions enter
the striated ducts which are lined by more columnar cells with many mitochondria.
Finally the saliva passes through the excretory ducts where the cell type is cuboidal
with stratified squamous epithelium (Shelton, 1996) 6 . The acinar cells first secrete
isotonic primary saliva and then the striated duct cells actively extract ions to render
the saliva progressively more hypotonic as it passes down the ducts towards the
mouth (Smith, 1996) 7 .
3.1 ) SALIVARY COMPOSITION AND ITS FUNCTIONS
Saliva is a dilute fluid, over 99% being made up of water. Whole saliva
collected from the mouth is a complex mixture. Apart from secretions of all the
glands it also contains desquamated oral epithelial cells, microorganisms and their
products, leucocytes, serum constituents, fluid from gingival crevice and food
remnants. The concentrations of dissolved solids (organic and inorganic) are
characterized by wide variation, both between individuals and within a single
individual. Of the approximately 750 ml of saliva secreted daily ,submandibular
5

glands account for 60%,parotid for about 30% and sublingual glands for 5% or less.
About 7% of saliva is derived from minor salivary glands 8 .
Salivary proteins
The proteins (organic component) of saliva comprise approximately
200mg per 100ml which is only about 3% of the protein concentration in plasma.
They include enzymes, immunoglobulins and other antibacterial factors, mucous
glycoproteins (mucins), traces of albumin, and certain polypeptides and
oligopeptides of importance in oral health. (Edgar, 1992) 9 .
Proline-rich proteins (PRP’s)
Human salivary PRP’s constitute a significant fraction of the total salivary
protein and have important biological activities (Hay et al, 1994) 10 . PRP’s are
inhibitors of calcium phosphate crystal growth. Almost all crystal growth inhibition
by PRP’s is due to the first 30 residues at the negatively charged amino-terminal end
of the molecule (Hay and Bowen, 1996) 11 . PRP’s are present in the initially formed
acquired pellicle, and have been reported to be present also in mature pellicles
(Lamkin et al, 1996) 12 . It has also been shown that PRP’s adsorbed onto
hydroxyapatite are strong promoters of adhesion for many common bacteria
(Gibbons and Hay, 1989; Li et al, 2001) 13,14 .Several salivary glycoproteins,
including the proline-rich glycoproteins and mucins, have lubricatory roles in
saliva, and the carbohydrate moieties of these molecules also affect their lubricating
properties (Aguirre et al., 1989) 15 .Salivary proline-rich proteins may act as defense
against tannins by forming complexes with them and thereby preventing their
6

interaction with other biological compounds and absorption from the intestinal tract
(Lu & Bennick, 1998) 16 .
Mucins
Human salivary mucins have a multifunctional role in the oral cavity in that
they lubricate oral surfaces, provide a protective barrier between underlying hard
and soft tissues and the external environment, and aid in mastication, speech and
swallowing (Tabak, 1995) 17 . The high-molecular- weight mucin (MG1) and the
low-molecular-weight mucin (MG2) have been isolated and characterized
biochemically as glycoproteins (Levine et al, 1987) 18 . Mucins have been intensively
studied, and much has been learned about their biochemical properties and their
interactions with oral micro-organisms and other salivary proteins (Offner and
Troxler, 2000) 19 .
Mucins tend to be asymmetrical molecules with an randomly organized
structure, consisting of a polypeptide backbone with carbohydrate side-chains. These
molecules are hydrophilic and entrain much water. MG1 and MG2 are the
predominant mucins in human saliva, providing lubrication and antimicrobial
protection for oral tissues (Baughan et al, 2000) 20 . MG1 is present in the mucous
acini of submandibular, sublingual, labial and palatine salivary glands (Nielsen et al,
1996) 21 . The site of MG2 synthesis is in mucous acini of both submandibular and
labial salivary glands (Cohen et al, 1991) 22 and in serous acini of submandibular,
sublingual, labial, and palatine salivary glands 21 .
These two major mucins create potential binding sites for microorganisms
at one of the major portals where infectious organisms enter the body (Thomsson et
7

al., 2002) 23 . Membrane-bound mucins are another class of mucin molecules which
exist both in secreted and membrane-bound forms 19 .
Histatins
Histatins comprise a group of small histidine-rich proteins present in the
saliva. The most significant function of histatins may be their anti-fungal activity
against Candida albicans (Tsai and Bobek, 1998; Koshlukova et al., 1999) 24, 25 .
Oral candidiasis may also modulate the levels of salivary histatin (Bercier et al,
1999; Jainkittivong et al, 1998) 26,27 . It has been suggested that histatins could be
used as components of artificial saliva for patients with salivary dysfunction 24 .
Histatins have been shown to be tannin-binding proteins in human saliva (Yan and
Bennick, 1995) 28 . Histatins also bind to enamel surfaces and hydroxyapatite in a
complex manner. Agglutinins
Salivary agglutinins are glycoproteins which have the capacity to interact
with unattached bacteria, resulting in clumping of bacteria into large aggregates
which are more easily flushed away by saliva and swallowed (Tenovuo & Lagerlöf,
1994) 29 . Bacterial binding to salivary proteins may in part account for individual
differences in the colonization of tooth surfaces. Agglutinins induce the aggregation
and clearance of streptococci from the oral cavity and are also important modulators
of initial plaque formation (Carlen et al, 1998) 30 . On the other hand, it seems that
salivary agglutinins may mediate the adherence of various bacterial species to the
tooth surfaces (Lamont et al., 1991; Stenudd et al., 2001) 31,32 .A number of salivary
proteins with an agglutinating capacity have been identified: parotid saliva
glycoproteins, mucins, sIgA, 2-microglobulin, fibronectin and lysozyme 32 .
8

Other polypeptides
Statherin is a small phosphoprotein (12,000 Da) relatively rich in tyrosine
and proline, which has the property of inhibiting hydroxyapatite crystal growth. It
also prevents the precipitation of calcium phosphates from supersaturated solutions
and may inhibit calculus formation. Sialin, a tetrapeptide can be utilized by several
bacteria, leading to formation of alkaline end products(amines)which are believed to
help regulate the plaque pH 9 .Minute amounts of albumin is present in the saliva as a
serum ultrafiltrate.Less than 100mg/l of albumin is found in saliva 33 .
Salivary enzymes
Amylase is one of the most important salivary digestive enzymes. It
consists of two families of isoenzymes, of which one set is glycosylated and the
other contains no carbohydrate (Makinen, 1989) 34 . Salivary amylase is a calcium
metalloenzyme which hydrolyses the alpha bonds of starches, such as amylose and
amylopectin 11 . Maltose is the major end-product.
It has been suggested that amylase accounts for 40 to 50% of the total
salivary gland-produced protein, most of the enzyme being synthesized in the
parotid gland. Human parotid saliva and submandibular saliva contain about 45 mg
and 30 mg of amylase, respectively, per 100 mg of protein 34 . However, it has also
been claimed that amylase makes up about 1/3 of the total protein content in parotid
saliva, and the content in whole saliva would be lower (Pedersen et al., 2002) 35 .
The concentration of amylase increases with the salivary flow rate, and it is
generally considered to be a reliable marker of serous cell function (Almståhl et al.,
2001) 36 .
9

In addition to its well-known function as a digestive enzyme, amylase has
been reported to act as an antimicrobial enzyme. Amylase activity exists also in
tears, nasal and bronchial secretions, milk, serum, urine and in the secretions of the
urogenital tract (Tenovuo, 1989) 37 . Amylase also interacts specifically with certain
oral bacteria and may play a role in modulating the adhesion of those species to teeth
(Scannapieco et al., 1993) 38 . It has been found that salivary amylase inhibits the
growth of Legionella pneumophila and Neisseria gonorrhea 37 . Amylase is also
present in human acquired pellicle in vivo (Yao et al., 2001) 39 . Fasting has been
found to decrease whole saliva amylase levels and activity. The amylase
concentrations in radiation-induced hyposalivation has been found to be reduced 36 .
Salivary lipase is another enzyme secreted by the lingual serous glands. It
plays a significant role in fat digestion in the newborn 36 .
Salivary Antimicrobial Proteins
Salivary immunoglobulins
Secretory IgA is the predominant one at approximately 20mg/100ml,
with IgG (1.5mg/100ml) and IgM(0.2mg/100ml)present in low amounts, possibly
arising from gingival crevice. Salivary secretory immunoglobulins (sIgA and sIgM)
originate from immune cells which are in the salivary glands, and are produced as a
host response to an antigenic stimulus (Brandtzaeg, 1989) 40 . The immunoglobulins
may be directed at specific bacterial molecules, including cell surface molecules
such as adhesins, or against enzymes. By binding to such molecules, adhesion of
specific bacteria to oral surfaces may be blocked, so preventing colonisation by the
10

affected species (Hay and Bowen, 1996, Zee et al, 2001) 11, 41 . Several studies have
confirmed that sIgA is mainly dimeric rather than monomeric, and it is associated
with an epithelial glycoprotein called SC (secretory component) (Seidel et al, 2001)
42 . At least 95% of the IgA normally appearing in saliva is produced by the local
gland-associated immunocytes rather than being derived from the serum 40 .
As the first line of defense against microbial invasion, sIgA is the
dominant immunoglobulin on all mucosal surfaces (Proctor and Carpenter, 2001) 43 .
High levels of sIgA are found in saliva of newborn infants, indicating the existence
of a competent oral mucosal immune system as early as within the first 10 days of
life (Seidel et al., 2000) 42 . It has been found that chewing stimulates epithelial cell
transcytosis of IgA and increases secretion of secretory IgA into saliva 43 . Salivary
levels of IgA have been widely studied, in healthy and also in diseased patients .In
HIV infected patients the IgA levels were higher than in healthy non-infected
controls (Mellanen et al, 2001) 44 .
Secretory IgM (sIgM) is not as resistant to proteolytic degradation as
sIgA. sIgM levels have been shown to be increased in infancy and in selective IgA
deficiency. Salivary IgG reaches the oral cavity by leakage through various epithelia
and is mainly added to whole saliva via crevicular fluid. It is mainly derived from
serum, although a minor fraction of the crevicular IgG may originate in local plasma
cells when the gingivae are inflamed 45 .
Traces of IgD probably reach whole saliva by passive diffusion like IgG.
IgD cannot be detected regularly in parotid fluid from normal adults but it appears
in whole saliva when present in serum. Salivary IgE most likely reaches external
11

secretions by passive diffusion, although a contribution from intraepithelial mast
cells in atopic and allergic individuals (Vernejoux et al, 1994) 45 .The biological
significance of the traces of IgE found in whole saliva is unknown 45 .
Nonimmunoglobulin proteins (Antibacterial proteins)
Lysozyme
Lysozyme represents the main enzyme of the nonspecific salivary
immune defense (Meyer and Zechel, 2001) 46 , and it is secreted mainly by the
submandibular and sublingual glands (Noble, 2000) 47 . Salivary lysozyme
hydrolyses specific bonds in exposed bacterial cell walls, causing cell lysis and
death.Lysozyme has been proposed as a lytic factor for bacteria to which
immunoglobulins have bound, mimicking in some respects the complement system
in serum.Lysozyme aggregates some bacterial species (Hay and Bowen, 1996) 11 .
Peroxidase systems
Peroxidases, salivary peroxidase and myeloperoxidase, catalyze a reaction
involved in the inhibition of bacterial growth and metabolism, and the prevention of
hydrogen peroxide (produced by the bacteria) accumulation, thus protecting proteins
from the action of oxygen and reactive oxygen species (Salvolini et al., 2000;Battino
et al., 2002) 48,49 . Salivary peroxidase catalyses the oxidation of thiocyanate ion
(SCN¯) which generate oxidation products that inhibit the growth and metabolism
of many microorganisms (Battino et al, 2002) 49 .
Lactoferrin
12

Lactoferrin is present in plasma and in mucosal secretions (van der Strate
et al., 1999) 50 . Salivary lactoferrin has antibacterial activity. Lactoferrin binds iron,
making it unavailable for microbial use (Tenovuo, 1989) 37 . Lactoferrin, in its
unbound state, also has a direct bactericidal effect on some microorganisms
including Streptococcus mutans strains 37 .
Other Organic compounds
Many free amino acids are present at low concentrations (below
0.1mg/100ml).When saliva is used by some oral bacteria as a sole source of nutrient,
amino acid content is too low to provide a rich growth medium. Urea is present at
levels about 12-20mg/100ml.It is hydrolysed by many bacteria with release of
ammonia, leading to rise in pH.Glucose (0.5-1 mg/100ml), vitamins, hormones and
clotting factors(factors VIII-XII) are also present.
Hormones present in saliva are cortisol, peptide hormones and growth
hormone. Studies suggest that salivary cortisol levels may be lower than the plasma
free level by as much as 20% (Read, 1989) 51 . Some investigators have found that
salivary cortisol is a better measure of adrenal cortical function than serum cortisol
and is particularly useful in studies with children and also to follow the response to
the ACTH (adrenocorticotropin hormone) test. Salivary determinations may also be
used to elucidate the role of cortisol in stress 31 .
Peptide hormones in saliva - It has also been suggested that proteins and
even small peptides could occur in saliva only as a result of contamination by
gingival fluid or plasma exudates. Such secretion is energy dependent, and it is not
clear that the salivary concentration of any protein secreted by such a mechanism
13

would bear any direct relationship to the circulating plasma concentration over short
periods of time 51 . However, there are published data on many peptide hormones in
saliva: human choriongonadotrophin, carcinoembryonic antigen, gonadotropins,
prolactin, thyroxine, melatonin, insulin (Lac, 2001) 52 and gastrin .Only minor
amounts of GH have been detected in human saliva (Rantonen et al., 2000) 53 .
Inorganic constituents
The major ions like sodium (0-80 mg/100ml), potassium (60-100
mg/100ml), chloride (50-100 mg/100ml) and bicarbonate (0-40 mg/100ml) are the
main contributors to the osmolarity of saliva, which is approximately half that of
plasma. Bicarbonate is also principal buffer in saliva. The Fluoride content (0.01-
0.04parts/10 6 ) is approximately similar to that of plasma, but is elevated slightly in
those who drink fluoridated water or use fluoridated toothpaste. These small
elevations in salivary levels are believed to be important in the anticaries action of
fluorides. Calcium (2-11 mg/100ml) and phosphate (6-71 mg/100ml) are present in
saliva partly bound to protein and partly in soluble complexes with carbonate,
phosphate or lactate. About 10% of the phosphate is in ester form, mainly in
phosphoproteins, but traces of pyrophosphate are also present 9 .
3.2) SALIVARY ALBUMIN
Albumin is the most abundant serum protein, accounting for more than
50% of all plasma proteins. Functions of albumin includes distribution of
extracellular fluid, regulation of osmotic pressure, acts as a transport agent for a
wide variety of substances like hormones, lipids, vitamins etc. Its molecular mass is
14

69 kDa and the normal serum reference limits are 40 - 52 mg/l. Albumin is
synthesized exclusively in the liver at a rate of 100 - 200 mg/kg/day. Factors that
regulate albumin synthesis are nutrition, hormonal balance and osmotic pressure.
The half life of albumin is approximately 15 - 20 days. About 4% of albumin is
degraded per day, but synthesis can be increased by as much as 100% by conditions
that decrease serum albumin or lower intravascular osmotic pressure (Weisiger,
1996) 54 . Increased levels are seen in dehydration. Decreased levels are seen in liver
diseases (Hepatitis, Cirrhosis), malnutrition, kidney disorders, increased fluid loss
during extensive burns and malabsorption (Doumasa et al, 1971) 55 . Nephrotic
syndrome is the best known example of systemic disorder with characteristic
proteinuria and subsequent hypoalbuminaemia which leads to oedema (Appel,
1996) 56 .
In the oral cavity, albumin is regarded as a serum ultrafiltrate to the
mouth (Oppenheim, 1970) 4 and it may also diffuse into the mucosal secretions
(Schenkels et al, 1995) 57 .There is about 3000mg/l of protein in human saliva of
which less than 100mg/l is albumin. This concentration shows wide variations
among individuals and can reach an average of 700mg/l in those with severe
periodontal inflammation 33 .The possible sulcular origin of salivary albumin was
suspected some time ago 4 ,with a lower content of albumin in glandular saliva than
in saliva collected from the floor of the mouth.Shielding the teeth and marginal
gingival with acrylic plates produced a significant decrease in albumin concentration
in whole saliva. Saliva from totally edentulous patients contained 5-6 times less
albumin than saliva from dentate individuals from the same age range, confirming
15

the sulcular origin for albumin 58 . Terrapon et al. (1996) 58 also found that the low
salivary albumin of old edentulous people was similar to that in a group of younger
individuals with a healthy periodontium 58 .
Salivary albumin is selectively adsorbed by different materials in the oral
cavity, which may enable the attachment of specific bacteria and thus alter the
composition of dental plaque (Kohavi et al., 1997) 59 . Salivary albumin has been
shown to increase in medically compromised patients whose general condition gets
worse (Meurman et al., 2002) 60 . Immunosuppression, radiotherapy, and diabetes are
examples of states where high concentrations of salivary albumin have been detected
(Tsutzu et al., 1981; Ben-Aryeh et al., 1993; Henskens et al., 1993; Meurman et al.,
1994; Mellanen et al., 2001) 5,61,33,44,62 . Salivary albumin levels have been used as a
marker for the degree of mucositis and inflammation in salivary glands 4 .
Panu et al(2000) 63 investigated the within subject variation of correlations
and concentrations between lysozyme, IgA, IgG, IgM, albumin ,amylase and total
protein in stimulated whole saliva of healthy adults in the course of 12 hour period.
Total protein correlated significantly with amylase, albumin and IgA through
different samplings. Butler et al. (1990) 64 found that albumin levels in whole saliva
fluctuated in most of the elderly patients in their study. Cuida et al. (1997) 65 found
that albumin concentrations were higher in both parotid and whole saliva in patients
with primary Sjögren’s syndrome (SS) than in the control group. However, the
output/min of albumin was lower in SS patients. It may be hypothesized that
salivary albumin can be used to assess the integrity of mucosal function in the mouth
(Meurman et al., 1997) 66 . In periodontitis patients, significantly increased levels of
16

salivary albumin have been reported 33 , and a significant correlation between
salivary albumin and gingival index in diabetic patients has been found 61 .
On the other hand, Sweeney and coworkers (1994) 67 did not find any
difference in serum albumin concentrations in elderly patients with mucosal
pathology in the mouth when compared with those with healthy mouths. In a study
Yoshihara et al. (2003) 68 found that there is a relationship between root caries and
serum albumin concentrations in elderly subjects 68 .
3.3) pH AND BUFFERING CAPACITY OF SALIVA
The hydrogen-ion concentration or pH is a measure of the acidity or
alkalinity of a solution. It is expressed as follows
1
pH = log 10____
(H +)
(H +) is the hydrogen-ion concentration of the solution in moles per litre.
The pH of a solution is defined as the negative logarithm of the hydrogen-ion
concentration, in an aqueous solution .At given temperature, in an aqueous solution,
the product of Hydrogen - ion concentration and Hydroxyl- ion concentration is
constant. The pH scale is introduced to avoid awkward small numbers 69 .
Saliva has a pH normal range of 6.2-7.6 with 6.7 being the average pH.
Resting pH of mouth does not fall below 6.3. In the oral cavity, the pH is maintained
near neutrality (6.7 to 7.3) by saliva. The saliva contributes to maintenance of the pH
by two mechanisms. First, the flow of saliva eliminates carbohydrates that could be
metabolized by bacteria and removes acids produced by bacteria. Second, acidity
17

from drinks and foods, as well as from bacterial activity, is neutralized by the
buffering activity of saliva. Buffering capacity can be defined as number of
equivalents of strong alkali or strong acid required to be added to a liter of the buffer
solution so as to change it’s pH by 1. Salivary buffering capacity is important in
maintaining a pH level in saliva and plaque. The buffer capacity of unstimulated and
stimulated whole saliva involves three major buffer systems (Bardow et al., 2000) 70 .
The most important buffering system in saliva is the carbonic acid /
bicarbonate system. Its concentration varies from less than 1mmol/l in unstimulated
parotid saliva to almost 60 mmol/l at very high flow rates, with whole saliva elicited
by chewing gum having a bicarbonate concentration of about 15 mmol/l.Thus, in
unstimulated saliva, the level of bicarbonate ions is too low to be an effective buffer.
The dynamics of this system is complicated by the fact that it involves the gas
carbon dioxide dissolved in the saliva 29 . The complete simplified equilibrium is as
follows:
CO2 + H2O - H2CO3 - HCO3¯ + H +
The increased carbonic acid concentration will cause more carbon dioxide to escape
from the saliva. The saliva bicarbonate increases the pH and buffer capacity of
saliva, especially during stimulation 70 . Henderson-Hasselbalch equation relates pH,
pKa and the ratio of salt concentration to undissociated acid. The relationship of the
pH and the bicarbonate concentration is given by the Henderson-Hasselbalch
equation as,
pH = pKa + log (HCO3¯)/(H2CO3)
18

in which the pKa (about6.1) is dissociation constant (ratio of the concentrations of
the dissociated ions and the undissociated acid) and H2CO3 which is about
1.2mmol/l are virtually independent of the flow rate. At very low flow rates, pH can
be as low as 5.3, rising to 7.8 at very high parotid flow rates. Individuals with
xerostomia will thus have a low salivary pH and a low salivary buffering capacity
because of the low bicarbonate concentration 9 .Concentration of Carbonic acid stays
remarkably constant at about 1.3mMol/L ,whereas pH and bicarbonate concentration
do change. As the rate of saliva production increases the more bicarbonate ion is
produced as a byproduct of cellular
metabolism. Carbonic anhydrase secreted by serous acinar cells of the parotid and
submandibular glands, drives the reaction converting carbonic acid to carbon
dioxide and water 9 .
The second buffering system is the phosphate system, which contributes
to some extent to the buffer capacity at low flow rate. The mechanism for the
buffering action of inorganic phosphate is due to the ability of the secondary
phosphate ion, HPO4¯, to bind a hydrogen ion and form an H2PO4¯ ion 9 .
The third buffering system is the protein system. In the low range of
pH the buffering capacity of saliva is due to the macromolecules (proteins)
containing H-binding sites 29 .The concentration of protein in saliva is only about one
thirtieth of that in plasma, so that too few amino acids are present to have a
significant buffering effect at the usual pH of the oral cavity.
The bicarbonate concentration is strongly dependent on secretion rate
(Birkhed and Heitze, 1989) 71 . Since bicarbonate is the chief determinant of the
19

uffer capacity, there is an interrelationship between pH, secretion rate and salivary
buffering capacity. Various methods have been used to measure the salivary buffer
capacity, including titration under oil, titration while open to air and titration with
CO2. Values obtained for buffer capacity in different studies are not comparable.
However, final pH under 3.5 for unstimulated saliva and 4.0 for stimulated saliva are
considered low.
From a practical point of view, the Dentobuff method has been developed
to assess the buffering capacity in dental practice. Based on the color change of the
indicator paper, the buffering capacity is assessed in comparison with a color chart.
The Dentobuff method to assess the salivary buffering capacities has been shown to
be valid (Ericson and Bratthall, 1989) 72 .
Low buffering capacity of saliva seems to reflect systemic acidosis and
may, consequently, be a sign of a worsening medical condition (Laine et al., 1992)
73 . However, the most important factor affecting salivary buffering capacity is
salivary flow rate. Bacteria are scavengers that attack the food particles in the oral
cavity to cause gingivitis. The pH in the blood and saliva of the oral cavity elevates
to above 7.6. Plaque bacteria take calcium compounds in the environment and use
the minerals to protect them from the high pH. The two key factors to plaque
formation is first there must be oral bacteria to attack food particles and elevate the
pH. Second the pH must elevate above 7.6 to grow dental plaque crystals that cause
periodontal disease 74 .
The parameters related to an intraoral mineralization tendency in
periodontitis-affected (P+) and periodontitis-free (P-) study subjects (16 adults, 46-
20

74 yrs, matched for sex and age) were compared (Sewon L et al, 1990) 75 . No
differences were found in the wet weight of plaque and in the flow rate, buffering
capacity of saliva between the groups. The subgingival area is bathed by gingival
fluid and is not controlled by the salivary buffering activity. The pH in the gingival
crevice may vary between 7.5 and 8.5, while the crevicular fluid ranges from pH
7.5 to 7.9. An alkaline pH in gingival crevices and periodontal pockets may exert a
selective force towards the colonization of periodontopathogens (Hamilton et
al,1989) 76 . Gingival crevicular pH has been considered an indicator of periodontal
health status and studied since 1954. Many investigators have measured the
crevicular pH and reported a relationship between pH and periodontal disease. Kosei
Kobayashi et al (1998) 77 investigated the fluctuation of GCF pH during
experimentally evoked gingivitis and occlusal trauma, to examine relationship
between pH and periodontal health status.The data suggested that the crevicular pH
level may not be influenced by experimental occlusal trauma,but shifts towards
alkaline with experimental gingivitis. Galgut P N (1995) 78 conducted a study to
investigate any possible correlations between pH and gingivitis & periodontal
pockets. Correlations between pH and gingivitis were not identified, but significant
correlations between pH and periodontal pockets were evident.Different pH readings
within a single pocket may imply that disease and reparative processes are occurring
simultaneously. Salivary pH showed no correlation with that in the periodontal
pockets (Watanabe et al, 1996) 79 .
Hong-Seop Kho(1999) 80 et al in his study investigated pH changes in
patients with end stage renal disease undergoing hemodialysis. Unstimulated whole
21

saliva showed high pH which was the result of a higher concentration of ammonia
due to ureal hydrolysis 80 .
3.4) SALIVARY FLOW RATE
Diminished salivary output can have deleterious effects on oral and
systemic health (Navazesh et al., 1992; Atkinson and Wu, 1994) 81,82 . Unstimulated
whole saliva is the mixture of secretions which enter the mouth in the absence of
exogenous stimuli such as chewing. Several studies of unstimulated saliva flow rates
in healthy individuals have found the average value for whole saliva to be about 0.3
ml/min.. Values below 0.1 ml/min are considered as hyposalivation, and values
between 0.1-0.25 ml/min as low 29 . Widely accepted normal values for stimulated
flow rates are 1.0 - 3.0 ml/min. The normal range is very large and includes
individuals with very low flow rates who do not complain of a dry mouth (Ship et
al,1991; Dawes, 1996) 83,84 . There is significant difference between genders in
unstimulated flow rates 84 .
Xerostomia (dry mouth) is a subjective feeling of oral dryness. It is
generally accompanied by salivary gland hypofunction and a severe reduction in the
secretion of unstimulated whole saliva (Sreebny, 1989) 85 , but xerostomia is not
necessarily reflected in the actually measured flow rates 85 .
Unstimulated saliva is usually collected with the patient sitting quietly,
with the head bent down and mouth open to allow the saliva to drip from the lower
lip into a sampling tube (the so-called draining method). The other most commonly
22

used technique for measuring unstimulated saliva is the spitting method where the
patient can spit out the saliva at regular intervals, while swallowing is inhibited.
Suction method and swab method also can be used 71 .The factors affecting
unstimulated saliva flow rate are degree of hydration, body position, exposure to
light, previous stimulation, circadian rhythms and drugs. Less important factors are
age, body weight, psychic effects, and functional stimulation (Dawes, 1987) 86 .
The amount of saliva in the mouth is not constant and varies within a
person over time and between individuals (Ship et al., 1991) 83 . Variation in
individual flow rates can be as high as 50% over a 24-hour period due to circadian
rhythms (Ferguson and Botchway, 1979) 87 and have been reported to exceed 50% in
cross-sectional healthy population studies (Ship et al., 1991) 83 . Normal variations
have been shown to be age and gender independent (Fischer and Ship, 1999) 88 .
Several studies have been made to evaluate the role of ageing in salivary
flow. Basically, there seems to be no age-related decrease in salivary flow rates
(Baum, 1981; Parvinen and Larmas, 1982; Thorselius et al., 1988) 89,90,91 , but
medication is one of the main factors causing reduced salivary flow (Strahl at al,
1990) 92 mainly in the elderly (Närhi et al., 1992) 93 . However, diminished resting
salivary flow in unmedicated healthy elderly subjects has been found in one study
(Percival et al., 1994) 94 . Many investigators have attempted to establish normal
ranges or “cut-off” values to distinguish normal from abnormal salivary function 2 .
A value of 0.1 ml/min has been suggested as the lower limit of normal unstimulated
whole saliva output (Sreebny and Valdini, 1988) 95 .
23

On the other hand, it has been shown in one study that healthy persons
in the lowest 10 th percentile of major salivary gland flow rates had oral health
similar to that of those in the highest 10 th percentile 83 . Single measurement of
salivary flow rate may be insufficient to determine how much saliva is necessary to
maintain oral health in particular individual 83,86 .
There are multiple causes of salivary hypofunction, including oral
disorders, systemic diseases, prescription and non-prescription medications,
chemotherapy, head and neck radiotherapy, psychogenic factors and decreased
mastication (Sreebny 1989; Sreebny and Schwartz, 1997; Ship et al., 1999; Ghezzi
et al., 2000) 2,95,96 . The most common cause of salivary gland hypofunction is the
intake of medicaments, over four hundred of which possess the ability to diminish
the flow of saliva. These have been identified and listed thoroughly in “Reference
guide to drugs and dry mouth” 96 . The feeling of dryness increases with the number
of drugs taken per day, but drugs usually do not cause permanent damage to the
structure of the salivary glands (Sreebny, 1989) 95 . Clinically, the most important
classes of drugs that continuously diminish the flow of saliva are antidepressants,
anticholinergics, diuretics and antihypertensive agents, and psychopharmaca
(Parvinen et al., 1984; Strahl et al., 1990) 90,92 .
3.5) SALIVARY CHANGES WITH AGEING
There is a continuing interest in understanding the influence of ageing
on physiologic processes. The goals of these efforts are to define which processes
change and which ones remain stable as a consequence of passage of time.
24

In 1969 Busse described ageing by distinguishing two pathways by
which overall functional status in an elder may occur. Primary ageing was defined as
the influence of the passage of time on a person, independent of extrinsic influences
or disabilities including stress, trauma or disease. The intent of this definition was to
delineate“pure ageing” phenomena. The other pathway, secondary ageing, was
defined as growing old in the presence of external influences. Although these
definitions have been conceptually useful, it is not clear if such distinctions should
actually be made at the level of an organ or organism. Earlier studies of ageing
frequently compared medically compromised older persons with healthier younger
ones and inappropriately concluded that physiologic function in many organ systems
was altered as the result of ageing (Jonathan A. Ship et al,1993) 97 .
Jonathan A. Ship et al (1993) 97 have come to two conclusions following
their study where they evaluated whether primary and secondary aging definitions
could be used to discriminate functional status of oral cavity. First one was that there
is little substantive difference in overall function and health of oral cavity when
described in terms of primary and secondary aging of human organism. Second one
was that the use of such broad definitions of ageing in an organism does not
necessarily lead to meaningful predictions of the health and function of an individual
organ system. Ageing, although considered a universal phenomenon, is not uniform
process across all the physiologic systems 97 .
Certain medical conditions, usually those associated with host defense
mechanisms and blood dyscrasias, can predispose to oral diseases such as
periodontal diseases. It is possible that oral diseases, especially those that affect the
25

teeth, could predispose to systemic problems. Aspiration pneumonia, a leading cause
of death among the frail elderly, can be associated with a history of periodontal
diseases. The combination of dental caries and periodontal diseases can be
associated with acute myocardial infarction and cerebral vascular accidents. Poor
oral health may be an important contributing factor in the development of significant
involuntary weight loss among the frail elderly. Elderly persons who complain of
xerostomia have exhibited a significant protein and calorie deficiency when
compared with age and gender matched persons with no complaints. Many reports
indicate that dependent living elderly have more dental morbidity, that is, fewer
teeth and more decay and periodontal disease than independent living elderly. The
medically healthy persons had excellent dental health whereas the sickest persons
were either edentulous or had many missing teeth (Walter J et al,1995) 98 .
Study conducted by MacEntee et al(1993) 99 demonstrates that the age
and gender of independent elders have very little direct influence on the oral health
or oral health related behaviour established early in life. Coming to the salivary
changes the elderly are particularly liable to oral dryness as a result of systemic
diseases and the use of many drugs. No change in parotid and sublingual flow is
noted but decrease in submandibular flow has been reported. Recently Narhi et al
(1992) 93 concluded in a study of subjects older than 76 years that actually
medication, not age, was the reason for reduced flow rate 93 .
Study conducted by Hanna Pajukoski et al (1997) 100 showed that
salivary flow rate and pH buffering capacity among the oldest and most frail patients
were lower than in younger patients with better general health. The drugs patients
26

used daily seemed to be principal cause of reduced salivary flow 100 .Resting and
stimulated whole saliva secretion rates were compared in old and young healthy
volunteers. The stimulated secretion rate was similar for both but the resting flow
rate was significantly lower in old females and males as compared with rates in the
young (Ben Aryah, 1984) 101 .
Salivary IgA and IgM values were increased in oldest age group
(85+).The IgM in the saliva mostly are derived from serum, thus dentate subjects
with a high prevalence of periodontal inflammation may show higher
concentrations. This also seems to the most probable explanation for the rise of
serum ultrafiltrates like
IgG, urea and albumin in the saliva of these patients. Infections appeared to cause
high salivary albumin concentration but are known to decrease serum albumin
concentrations. Increased albumin concentrations are also noted in connection with
dehydration 100 .Salivary albumin in edentulous patients were similar to younger
individuals with healthy periodontum 58 .Salivary amylase which is a marker of
exocrine function of salivary glands is seen in slightly higher concentration in
saliva’s of edentulous patients 100 . Sweeney MP et al (1994) 67 assessed the oral
health, oral microbiology and micronutrient status of geriatric patients. Those with
mucosal pathology had lower serum iron concentrations. Albumin concentration
tended to be below the lower limit of the reference interval 67 .
3.6) SALIVARY CHANGES IN GINGIVITIS AND PERIODONTITIS.
27

Gingiva is that part of the oral mucosa that covers the alveolar
processes of the jaws and surrounds the necks of the teeth. In clinically healthy
gingiva of humans, sulcus depth of 1.8 mm with variations from 0-6 mm is reported
.The gingival sulcus contains a fluid that seeps into it from the gingival connective
tissue through the thin sulcular epithelium and is called as GCF (gingival crevicular
fluid).Periodontal ligament is the connective tissue that surrounds the root and
connects it to the bone. It is continuous with the connective tissue of the gingiva
and communicates with the marrow spaces through vascular channels in the bone.
Inflammation of the gingival tissue results in gingivitis, which if not resolved leads
to inflammation of the periodontium called as periodontitis 102 .
Sequence of events in the development of gingivitis is in 3 stages
namely Initial, Early and Established lesions. In the Initial lesion clinically no
change is apparent. Vascular changes take place leading to exudation of fluid from
the gingival sulcus.If this phase is not resolved then stage II gingivitis (Early) sets
in, which is usually seen in 4-7 days and is accompanied by vascular proliferation
and chronic inflammatory cell infiltrate. Clinically erythema and bleeding on
probing are seen. Stage III is an established lesion, seen in 14-21 days and is an
advanced stage of the early lesion with continued loss of the collagen fibre bundles.
Stage IV is an advanced lesion characterized by the extension of the lesion into
alveolar bone leading to phase of periodontal breakdown 102 .
Periodontitis involves the destruction of the connective tissue attachment and
the adjacent alveolar bone. In periodontitis, the gingival crevice is deepened to form
a periodontal pocket due to the apical migration of the junctional epithelium along
28

the root surface. The induction and progression of periodontal tissue destruction is a
complex process involving plaque accumulation, release of bacterial substances and
host inflammatory response .It is characterized by pocket formation and bone loss.
Pockets are caused by microorganisms and their products, which produce
pathologic tissue changes that lead to deepening of the gingival
sulcus.Periodontitis, may be classified as slowly progressive periodontitis or adult
periodontitis, rapidly progressive periodontitis, necrotizing ulcerative periodontitis
and refractory periodontitis. Rapidly progressive periodontitis is further subdivided
into adult onset (more than 20 years) ,pubertal and adolescent onset (age between
11 and 19 years) and prepubertal onset (less than 11 years).It can also be classified
based on probing attachment loss which is 2-4mm in mild periodontitis, 4-7mm in
moderate periodontitis and more than 7 mm in severe periodontitis. It has been
classified based on disease activity and severity as acute or chronic and based on
distribution of lesions as localized or generalized 102 .
Gingivitis and periodontitis are oral diseases which are characterized by
chronic inflammation. It is generally accepted that oral bacteria cause inflammatory
responses, which can result in tissue destruction in various ways. In the first place
bacteria can directly contribute to periodontal disease by releasing proteolytic
enzymes which can damage the oral tissues. In addition, oral bacteria may induce
tissue destruction indirectly by activating host defence cells eg. polymorphonuclear
leucocytes (PMN’s),which can release their lysosomal proteolytic enzymes at the
inflamed sites((Henskens Y M C et al 1993) 33 . Periodontitis is a destructive disease
primarily related to chronic plaque accumulation. Putative periodontopathic
29

acteria such as Porphyromonas gingivalis, Prevotella intermedia or Actinobacillus
actinomycetemcomitans are suspected to play a role in the periodontal disease
process. They release proteolytic enzymes that degrade salivary
proteins,immunoglobulins and collagen type I 102 .
The heritability of salivary protein concentrations was investigated in
stored samples of clarified stimulated whole saliva from adult twins participating in
a study of periodontal disease genetics. Findings, taken as sibling correlations,
support a genetic contribution to saliva protein concentrations. Total protein also
showed a significant positive correlation with gingivitis (Rudney JD et al, 1994) 103 .
The presence of certain factors in the saliva or GCF can act as markers
in periodontal and gingival lesions. The lysosomal cysteine proteinases, cathepsins
B,H and L have been detected in gingival crevicular fluid of subjects with gingivitis
and periodontitis (Kunimatsu et al,1990) 104 .These cystatins can degrade collagen
and it has been suggested that they play a role in tissue destruction in periodontal
disease. Henskens YMC et al (1993) 33 assessed the salivary total protein, albumin
and cystatin concentrations in saliva of healthy subjects and of patients with
gingivitis or periodontitis. An increase in salivary cystatins, proteins and albumin
was noted in patients with gingivitis or periodontitis. GCF showed increased
albumin content in inflamed ginigival site when compared to pre-inflammatory
GCF (M.Bickel et al, 1985) 105 .Whereas, investigations carried on by Henskens et al
(1996) 106 showed that levels of whole saliva albumin and IgA, originating from
sources other than glandular cells were not different between healthy and
periodontitis subjects and were also not correlated with the typical salivary gland
30

proteins. Gelatinases /type IV collagenases in saliva and GCF were found in
periodontitis patients (Ingman T Sorca, 1994) 107 .
Saliva can be used as an indicator of prognosis during periodontal
treatment. Henskens et al (1996) 106 evaluated the effect of periodontal treatment on
the protein composition of whole and parotid saliva. Significant changes in salivary
protein composition including that of albumin occurred only in whole saliva, after
treatment. Concentrations of parotid Cystatin S was unchanged during the
periodontal treatment process 108 . Salivary levels of alpha 2-macroglobulin, C-
reactive protein, cathepsin G and elastase levels are directly related to an individual's
periodontal status (Pederson et al,1995) 109 .
Protein carbonyl concentrations were determined as an index of
oxidative injury in a sample of whole unstimulated saliva. Poor periodontal health
was associated with increased concentrations of protein carbonyls in saliva. Women
had significantly lower total antioxidant status than men, regardless of periodontal
health. Periodontal disease is associated with reduced salivary antioxidant status
and increased oxidative damage within the oral cavity (Sculley, 2003) 110 .
Fujikawa et al (1989) 111 studied correlation between the pH level and
the microflora in periodontal pockets in the various stages of periodontal disease. A
change in pH level was seen in deep pockets or severe gingival inflammation. A
close correlation was seen between salivary and crevicular pH. The pH level was
significantly positively related with the proportion of coccoid forms, but was
negatively correlated with the proportion of motile organisms that are reported to be
related with periodontal disease 111 .
31

3.7) SALIVA AS A DIAGNOSTIC FLUID
Salivary diagnosis is an increasingly important field in dentistry,
physiology, internal medicine, endocrinology, pediatrics, immunology, clinical
pathology, forensic medicine, psychology and sports medicine .A growing number
of drugs, hormones and antibodies can be reliably monitored in saliva, which is an
easily obtainable, non-invasive diagnostic medium (Mandel, 1990; Tabak, 2001)
1,112 . Thus, salivary diagnosis is anticipated to be particularly useful in cases where
repeated samples of body fluid are needed but where drawing blood is impractical,
unethical, or both. It is easy to collect, store and ship, obtained at low cost and is
collected relatively safely and non invasively than serum (Harold C.Slavkin, 1998)
113 . Salivary concentrations of drugs and hormones also represent the free fractions
of serum in many instances, with good correlations with the respective total
concentrations in serum (Hofman, 2001) 114 .
Examples of molecularly based determinants used in saliva fluid diagnostics
(Harold, 1998) 113
Detection of viruses using antibodies.(IgG,IgA,IgM) specific for a viral antigen.
Hepatitis A and B, HIV-1, 2, Measles, Mumps and rubella.
Detection of microbe-specific antigenic determinants.
• Neuraminidase(enzyme associated with Influenza virus)
32

• N-acetylglucosamine(molecule associated with streptococcus A)
• Salivary Estradiol hormone(preterm labor indicator)
• Epidermal growth factor, cathepsin-D and Waf 1(breast cancer markers)
• Zinc binding cystic fibrosis antigen (proposed cystic fibrosis biomarker)
• Glutamic acid decarboxylase autoantibody(marker for Type 1 Diabetes)
Detection of bacterial organisms in saliva
• Lactobacillus acidophilus
• Streptococcus mutans
• Porphyromonas gingivalis
Examples of chemicals and molecules identified and measurable in saliva
Aldosterone , Ethanol, Antipyrine ,Insulin,Caffeine, Lithium, Cannabinoids ,
Melatonin, Carbamazepine ,Opiates, Cocaine, Phenytoin, Cortisol , Progesterone
Cotinine ,Testosterone , Estradiol, Theophylline.
Multiple specimens of saliva for steroid hormone analysis can be easily
collected by the patient, at home, to monitor fertility cycles, menopausal
fluctuations, stress and other diurnal variations 114 .
Salivary antibody levels can be determined to screen for infectious
diseases. Anti-HIV antibody immunocapture assays have also been developed and
tested for saliva, which could be useful in high-risk groups under field conditions in
developing countries (Pasquier et al., 1997) 115 . Salivary assays have been used for
33

monitoring of Hepatitis A and B , measles, Epstein Barr virus, Rubella, Parvovirus
B 19, Human herpes virus 6, Helicobacter pylori and Rotavirus infection (Mandel,
1990; Madar et al., 2002) 1,116 . In addition to measuring antibody, it is possible to
identify a number of viral antigens in saliva, for example mumps and
cytomegalovirus. Saliva has also proven to be a convenient source of host and
microbial DNAs 17 .
There has been a growing interest in the use of saliva in pharmacokinetic
studies of drugs and in therapeutic drug monitoring in a variety of clinical situations.
It has been suggested that drug levels in saliva reflect the free, non-protein-bound
portion in plasma and hence may have a greater therapeutic implication than the
total blood levels 1 . Lipid solubility is a determining factor in salivary excretion of
drugs, and the degree of acidity and basicity of a drug will determine its
salivary/plasma ratio. The salivary flow rate, pH, sampling conditions,
contamination and many other pathophysiological factors may influence the
concentrations of drugs in saliva (Liu and Delgado, 1999) 117 . Drugs currently
monitored in saliva include anticonvulsants, theophylline, salicylate, digoxin, anti-
arrhythmic drugs, ethanol, benzodiazepines, amitryptyline, chlorpromazine,
methadone, marijuana, cocaine and caffeine 117 .
The manufacturer of the Orasure Oral Specimen Collection system says
that the creation of the device has resulted in the approval by FDA for qualitative
determination of 4 out of 5 drugs called NIDA-5(National Institute of Drug
Abuse).It includes approved assays for marijuana,cocaine,methamphetamine,and
opiates. The methodology also can determine the presence of cotinine.Using levels
34

of nicotine in air and cotinine in saliva scientists are able to calculate risk levels of
passive smoking exposure in workplace (Harold,1998) 113 .
Saliva has implication in the criminal justice field. In circumstances
where alcohol is involved, a dipstick is used to obtain saliva sample and an enzyme
based reaction takes place .Enzymatic oxidation of alcohol to acetaldehyde by
alcohol dehydrogenase takes place. A chromogen is used to indicate the level of
alcohol 113.
It has become apparent that many systemic diseases affect salivary gland
function and salivary composition. Primary Sjögren’s syndrome (SS) is a common
autoimmune disorder characterized by generalized dessication, exocrine
hypofunction and serologic abnormalities. More than 90% of the patients are
women, and one of the main diagnostic procedures is biopsy of the minor salivary
glands of the lip. It has been suggested that whole saliva flow rate and gland-specific
sialometry and sialochemistry (Kalk et al., 2002) 118 could be used to provisionally
diagnose SS. It has been suggested that diminished output of salivary defense
factors, rather than their absolute concentrations, may be related to the oral health
problems seen in SS patients. Cystic fibrosis affects all of the exocrine glands to
varying degrees (Ferguson, 1999) 119 . The most dramatic changes in the composition
of saliva reported have been an elevation in calcium (Ca) and proteins, and this
reduces the flow rate of minor salivary glands to virtually zero. Normally the flow
rate of single labial gland is 0.1 µl/min 119 . This phenomenon can be used as a
diagnostic test by measuring the flow from labial glands of the lower lip 1 .
35

The sodium (Na) and potassium (K) concentrations of saliva are markedly
affected by corticosteroids, especially aldosterone. The Na/K ratio of stimulated
whole saliva can be used in diagnosing and monitoring Cushing’s syndrome and
Addison’s disease. Investigators have also demonstrated the diagnostic value of
Na/K ratio in primary aldosteronism 113 .
In several clinical situations salivary analysis has provided valuable
information for both the clinician and the investigator. These situations include
digitalis toxicity, stomatitis in chemotherapy, specific secretory IgA deficiency,
smoking, ovulation time, relation of dietary factors to cancer and chronic pain
syndromes 1 .
Human saliva contains a large number of enzymes derived from the
salivary glands, oral microorganisms, crevicular fluid, epithelial cells, and other
sources. It has been difficult to standardize saliva collection methods and enzyme
analytical procedures so that direct comparisons between different laboratories
would be possible 34 .Interpretation of results has also proved to be difficult.
However, various studies have been made to find correlations between diseases or
clinical situations and salivary enzyme levels.
Saliva is essential for alimentation, remineralization of teeth, and the
protection and lubrication of oral mucosal tissues. Measurement of the patient’s
salivary flow is of primary importance in oral medicine and dentistry 2 . For many
years dental investigators have been exploring changes in salivary flow rate and
composition as a means of diagnosing and monitoring a number of oral diseases. It
has even been suggested that analysis of saliva may also offer a cost-effective
36

approach to the assessment of periodontal diseases in populations (Kaufman and
Lamster, 2000) 120 , even though no specific salivary marker of periodontal disease
activity has been found so far 120 .
Diagnostic tests in normal dental practice
Saliva is well adapted to protection against dental caries. The buffering
capacity of saliva, the ability of saliva to wash the tooth surfaces and to control
demineralization and mineralization, the antibacterial activity of saliva and perhaps
other mechanisms all contribute to its essential role in the health of the teeth.
Measurement of salivary flow is an invaluable diagnostic tool in determining the
prognosis of alternative treatment plans 92 . In modern dental practice, diagnostic
salivary measurements, at least salivary secretion rate and buffering capacity, should
be used to supplement the information and clinical findings with regard to
prevention of dental caries 29 .However, since carious lesions are the result of a
multifactorial disease, assessment of a few salivary factors is not sufficient unless
they are of overriding importance, which may occur in an individual patient 71 .
Salivary bacterial counts, for example streptococcus mutans and
lactobacillus dip slide tests, are widely used in clinical practice in caries risk
assessment. The current tests may be useful for estimating caries activity due to bad
dietary habits, and establishing the presence of infection and salivary yeasts for the
determination of the patient’s medical condition. However, these tests may be
limited in their applicability in the assessment of caries activity and in caries
37

prediction (Pinelli et al., 2001) 121 .Mutans streptococci are acidogenic and aciduric,
and can produce extracellular glucans and adhere to tooth surfaces. Several methods
are available to measure the levels of mutans streptococci in saliva and in plaque.
The so-called ´Strip Mutans test´ is based on the ability of mutans streptococci to
grow on hard surfaces, and it has been developed as chair-side test 121 .
Lactobacilli are associated with caries. They are more dependent on
retentive sites being available in high numbers, and hence lactobacillus counts have
been used to predict the increment of new caries lesions (Smith et al., 2001) 122 . The
standard laboratory method of determining the number of lactobacilli includes the
use of selective medium, Rogosa S L-agar. Chair-side methods for lactobacilli have
also been developed, since the ´Dentocult LB´ method in 1975 (Larmas, 1975) 123 .
Yeasts, mainly Candida albicans, are commensals of the oral cavity in
the majority of adult patients but many diseases may predispose to their
dissemination (Odds, 1988) 124 . Candidal colonization has been demonstrated in
periodontal pockets, refractory periodontitis and failing dental implants (Pizzo et al.,
2002) 125 . In a review by Odds (1988) 124 the prevalence of yeasts in saliva of healthy
persons was stated to be 37%. In denture wearers yeasts may be cultivated from 85%
of the subjects. A chairside method, `Oricult ®´, has been developed for detecting
yeasts (Nicherson agar) in the mucous membranes and saliva 124 .
Saliva can be used to detect Porphyromonas gingivalis.Increased ability
to detect minute mounts of DNA using polymerase chain reaction has led to a
molecularly based assay using saliva to detect P gingivalis.This pathogen is rarely
found in saliva samples obtained from periodontally healthy children and young
38

adults, but is frequently identified in the saliva obtained from adult patients with
periodontitis. A simple device that collects gingival fluid may improve molecularly
based assay. The clinician can further use it to monitor and measure improvement
and eventually prevention of the disease process 113 .
It has recently been shown that low secretion rate of saliva and the high
scores of lactobacilli and Streptococcus mutans have a significant influence on
complications of fixed metal ceramic bridge prostheses and this should be taken into
consideration in choosing patients for prosthetic treatment with fixed prosthodontics
(Näpänkangas et al., 2002) 126 . Since salivary flow and its composition are essential
in the protection and lubrication of oral mucosal tissues, salivary tests have also
significant predictive value in prosthodontic treatment planning. Successful
management of complete and removable partial dentures is complicated by a
reduction in salivary flow .It has been suggested that salivary tests should be
performed and analyzed before planning an extensive and expensive restorative
therapy or orthodontic treatment (Birkhed and Heintze, 1989) 71 and on routine basis
with geriatric patients (Strahl et al, 1990) 92 .
39

4.METHODOLOGY
Source of data
Department of Oral Medicine and Radiology & Department of Periodontics,
A.B.Shetty Memorial Institute of Dental Sciences.
Methodology
Patients were chosen from the department of Oral Medicine and Radiology
and department of Periodontics, A.B.Shetty Memorial Institute of Dental Sciences.
80 patients were chosen on the basis of presence of gingivitis and periodontitis under
2 different age groups.
Criteria for gingivitis was based on NIDR criteria
NIDR-Gingival inflammation Index (Bleeding index)
0= No bleeding
1= Bleeding after probe is placed in gingival sulcus upto 2 mm and drawn
along inner surface of the gingival sulcus.
Criteria for periodontitis was based on loss of attachment with pocket depth of >/= 5
mm in at least 8 sites.
First group comprised of 40 young patients between 20 and 35 years of
age with no systemic diseases and not on medication.Second group comprised of 40
40

elderly patients of 65 years and above, with no systemic diseases and not on
medication.20 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. (Annexure 1).Based on the above mentioned criteria patients were
subgrouped under 6 groups as:
1. Young - Control (Number of subjects = 20)
2. Young - Gingivitis (Number of subjects = 20)
3. Young - Periodontitis (Number of subjects = 20)
4. Elderly - Control (Number of subjects = 20)
5. Elderly - Gingivitis (Number of subjects = 20)
6. Elderly - Periodontitis. (Number of subjects = 20)
Human whole unstimulated saliva was collected by spitting method
without swallowing with the patient seated in an upright position between 11am and
12noon. Approximately 5 ml of saliva was collected. Flow rate was calculated as
volume collected divided by the time required for the collection. Salivary samples
were then labelled and estimated for salivary total protein, albumin concentrations,
pH and buffer capacity.
Salivary protein estimation
41

Salivary protein estimation was done based on Biuret method. Protein forms
a coloured complex with cupric ions in alkaline medium. Based on this principle
salivary protein estimation was done by mixing undiluted saliva with the reagent
provided and measuring the coloured product using a photoelectric colorimeter at a
wavelength of 536 nm. Details of the procedure is described in Annexure 3.
Salivary Albumin estimation
Salivary albumin was estimated using Bromocresol green method. The reaction
between albumin in saliva and the dye Bromocresol green produces change in colour
which is proportional to the albumin concentration in the saliva. It was estimated
using a photoelectric colorimeter at wavelength of 630 nm.Details of the procedure
is described in Annexure 2.
Salivary pH and buffering capacity
Salivary pH was estimated with the help of a pHmeter. Then, titration method was
used to determine the buffering capacity as described in Annexure 4.
42

STUDY DESIGN
SALIVARY TOTAL PROTEIN, ALBUMIN, pH & BUFFERING CAPACITY
GROUP 1
YOUNG
PATIENTS
20-35 YEARS
GROUP 2
ELDERLY
PATIENTS
65 YEARS AND
ABOVE
43
SUBGROUP 1
YOUNG
CONTROL
SUBGROUP 2
YOUNG
GINGIVITIS
SUBGROUP 3
YOUNG
PERIODONTITIS
SUBGROUP 4
ELDERLY
CONTROL
SUBGROUP 5
ELDERLY
GINGIVITIS

44
SUBGROUP 6
ELDERLY
PERIODONTITIS
Fig-1) ARMAMENTARIUM USED IN SALIVARY TOTAL
PROTEIN ESTIMATION
Fig -2) COLORIMETER

Fig -3) ARMAMENTARIUM USED IN SALIVARY ALBUMIN
ESTIMATION
Fig -4) pHMETER
45

RESULTS
Present study included 120 subjects, who were grouped based on their age as Young
and Elderly. Further subgroups were made as Controls, Gingivitis and periodontitis
subjects under each group. The values of the parameters that are salivary total protein,
salivary albumin, pH, buffer capacity, and flow rate of our study sample are tabulated
in tables 1-6 in Annexure 5.
The biochemical values of this study was 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.
Of 120 subjects, 61 were male and 59 females. Significance of parameters
like pH, buffer capacity, salivary total protein, salivary albumin and flow rate was
estimated among subgroups in young patients using Fisher’s test (Table 7).Salivary
Table 7-Comparison of parameters in subgroups –Young (Fisher’s test)
pH
B.CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOWRATE
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
N Mean Std. Deviation F
p
20 6.5800 .4686
20 6.5900 .3478
20 6.6800 .3995 .364 .697
20 5.8100 .3291
20 5.8850 .3017
20 6.0370 .2880 5.072 . 708
20 .8950 .2012
20 1.2400 .2909
20 1.6250 .4290 25.877 .001 vhs
20 .1020 .0535
20 .2400 .8208
20 .4150 .1137 65.537 .001 vhs
20 .5400 .1314
20 .5250 .1293
20 .5050 .1395 .346 .709
46

total protein & albumin estimation were shown to be very highly significant
(p=0.001) markers in the young group. Other parameters like pH, buffer capacity and
flow rate estimation were not significant as shown in table 7.
Table 8 shows comparative analysis of parameters among subgroups in
young patients using Tukey HSD test(ANOVA).Salivary total protein concentration
rise was highly significant (p=0.004) and very highly significant(p=0.001) in
gingivitis and periodontitis subgroups respectively when compared with the controls.
The value rise was very highly significant (p=0.001) when gingivitis subgroup was
correlated with periodontitis subgroup. Salivary albumin concentration rise was very
highly significant (p=0.001) when controls were correlated with gingivitis and
periodontitis subgroup and when gingivitis subgroup was correlated with periodontitis
subgroup. Other parameters like pH, buffer capacity and flow rate did not correlate
significantly in all the subgroups among the young.
Table 8 .Comparison of the parameters in different subgroups – Young
(Tukey HSD test)
Dependent Variable
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
(I) SUBGROUP
Control
Gingivitis
Control
Gingivitis
Control
Gingivitis
Control
Gingivitis
Control
Periodontitis
47
(J) SUBGROUP
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Gingivitis
Mean
Difference
(I-J)
-.1000
Sig.
.997
-.1000
-.0900
.720
.766
-.0650 .896
-.2270 .058
-.0680
-.3450
.764
0.004 hs
-.7300 .001 vhs
-.3850 .001 vhs
-.1380 .001 vhs
-.3130 .001 vhs
-.1750
-.0150
.001 vhs
.933
-.0350
-.0200
.686
.884

Among the elderly subjects salivary total protein and albumin values were shown
to be very highly significant (p=0.001, Fisher’s test) parameters as shown in Table
9.Estimation of other variables were not significant.
Table 9-Comparison of parameters in subgroups –Elderly (Fisher’s test)
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
All the parameters in three subgroups among the elderly were correlated using
Tukey HSD test as shown in table 10a and 10b. Salivary total protein concentration
rise was significant (p=0.023) and very highly significant (p=0.001) in gingivitis and
periodontitis subgroups respectively when compared with the controls. The value rise
was very highly significant (p=0.001) when gingivitis subgroup was correlated with
periodontitis subgroup for salivary protein as well as for albumin. pH (Table 10a) and
other parameters were not significant.
N Mean Std. Deviation F p
20 6.7400 .4031
20 6.6700 .4041
20 6.6200 .4046 4.625 .980
20 6.1050 .3233
20 5.9900 .1730
07
.923 .403
20 5.9200 .3150
20 .8350 .2368
20 1.1400 .1569 20.786 .001 vhs
20 1.5550 .5443
20 0.0865 .0142
20 .2550 .1050
20 .4650 .1348 73.352 .001 vhs
20 .3950 .1191
20 .3600 .1273 1.846 .167
20 .3200 .1240
48

Table 10a .Comparison of pH in different subgroups of
elderly group using Tukey HSD test.
CLASS
Old
(I) SUBGROUP
Control
Gingivitis
(J) SUBGROUP
Gingivitis
Periodontitis
Periodontitis
Mean
Difference
(I-J) p
.0700 .839
.3550 .126
.2850 .063
Table 10b .Comparison of the parameters in different subgroups –
Elderly (Tukey HSD test)
Dependent Variable
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
(I) SUBGROUP
Control
Gingivitis
Control
Gingivitis
Control
Gingivitis
Control
Gingivitis
(J) SUBGROUP
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Mean
Difference
(I-J) Sig.
-.4500 .491
.1300 .998
2.4650 .454
-.3050 0.023 sig
-.7200 .001 vhs
-.4150 .001 vhs
-.1685 .001 vhs
-.3785 .001 vhs
-.2100 .001 vhs
-.0350 .645
-.0750 .142
-.0400 .565
Table 11 and Graphs 2, 3 and 4 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 were 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 were significantly (p=0.054) higher than that of the young control group
(Graph 6). pH ,salivary total protein and albumin estimations did not show any
significant changes. Graph 5 and 6 correlates the pH and buffering capacity among
young and elderly in different subgroups.
49

Table 11.Correlation of the parameters in the subgroups among the young and
elderly (t-test).
SUBGROUP
Control
Gingivitis
Periodontitis
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
CLASS
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
Young
Old
N Mean
S.D
t
20 6.580 .4686 1.1580
20 6.740 .4031 p=.254 ns
20 5.810 .3291 1.9870
20 6.015 .3233 p=.054 sig
20 .8950 .2012 .8630
20 .8350 .2368 p=.393 ns
20 .1020 .0536 1.2510
20 .0865 .0142 p=.219 ns
20 .5400 .1314 3.6570
20 .3950 .1191 p=.001 vhsig
20 6.590 .3478 .6710
20 6.670 .4041 p=.656 nsig
20 5.885 .3017 .8980
20 5.990 .1117 p=.675 nsig
20 1.240 .2909 1.3530
20 1.140 .1569 p=.184nsig
20 .2400 .0821 .5030
20 .2550 .1050 p=.618 nsig
20 .5250 .1293 4.0670
20 .3600 .1273 p=..410 ns
20 6.680 .3995 2.4390
20 6.677 .3646 p=.019 sig
20 6.037 .2880 1.5930
20 5.920 .3150 p=.12 ns
20 1.625 .4290 .4520
20 1.555 .5443 p=.654 ns
20 .4150 .1137 1.2680
20 .4650 .1348 p=.213 ns
20 .5050 .1395 4.4340
20 .3200 .1240 p=.001 vhs
Table 12 and Graph 1 estimates the significance of different parameters in
combined young and elderly groups using Fisher’s test. Salivary total protein &
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 (S.D=0.21)g/ml, 1.19g/ml (S.D=0.23) & 1.59 g/ml(S.D=0.48). Total mean
50

salivary albumin for controls, gingivitis and periodontitis patients was
0.09(S.D=0.04), 0.24(S.D=0.09), and 0.44(S.D=0.12) mg/ml.
Table-12 Estimating significance of the parameters in the study.
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
Control
Gingivitis
Periodontitis
All the variables were compared among different subgroups in Table 13
using Tukey HSD test. Again salivary total protein & albumin rise was very
significant (p=0.001) when controls were compared with gingivitis and periodontitis
subgroup and gingivitis was correlated with periodontitis subgroup. Table 14
correlated the gender variations in the parameters among all the subgroups of the
young and elderly using t-test. 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 females (Graph 7).
N Mean Std. Deviation F p
40 6.6600 .4390
40 6.6300 .3743
40 6.5325 .4060 1.072 .346
40 5.9125 .3383
40 6.0325 .8838
40 5.9610 .3077 1.070 .346
40 .8650 .2190
40 1.1900 .2362
40 1.5900 .4851 46.674 .001 vhs
40 0.0942 .0394
40 .2475 .0933
40 .4400 .1257 138.182 .001 vhs
40 .4675 .1439
40 .4425 .1517
40 .4125 .1604 1.310 .274
51

Table 13. Comparing parameters among subgroups
Dependent Variable
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
CLASS
Young
Old
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
pH
BUFFER
CAPACITY
SALIVARY
TOTAL
PROTEIN
SALIVARY
ALBUMIN
FLOW RATE
(I) SUBGROUP
Control
Gingivitis
Control
Gingivitis
Control
Gingivitis
Control
Gingivitis
Control
Gingivitis
SEX
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
(J) SUBGROUP
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Gingivitis
Periodontitis
Periodontitis
Mean
Difference
(I-J)
. -0300
p
.942
.1275 .344
-.0975 .534
-1.3150 .404
-.O485 .999
1.2665 .431
-.3250 .001 vhs
-.7250 .001 vhs
-.4000 .001 vhs
-.1532 .001 vhs
-.3458 .001 vhs
-.1925 .001 vhs
-.0250 .743
-.0550 .243
-.0300 .653
Table 14. Correlating parameters in subgroups among males and females
N Mean Std. Deviation t
29 6.7586 .3551 2.7790
31 6.6839 .4067 p=.707 ns
29 6.0828 .2550 2.3460
31 6.0616 .3628 p=.062 ns
29 1.2724 .4061 .3250
31 1.2355 .4680 p=0.746 ns
29 .2490 .1597 .1620
31 .2555 .1525 p=0.872 ns
29 .6241 .0987 8.5080
31 .4290 .0782 p=.001 vhs
32 6.5719 .4467 .9830
28 6.6286 .3799 p=0.601 ns
32 5.6382 .8175 .9830
28 5.8750 .2927 p=.33 ns
32 1.2094 .4720 .5880
28 1.1393 .4475 p=0.559 ns
32 .3059 .2013 1.6960
28 .2264 .1548 p=.095 ns
32 .4344 .0865 6.5910
28 .2714 .1049 p=.001 vhs
52

BAR CHARTS
Graph. 1. Comparison of the variables among subgroups.
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Salivary total
protein
Salivary
albumin
Control
Gingivitis
Periodontitis
Flow rate
Graph .2. Comparing variables among young and elderly in Controls
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Salivary total
protein
Salivary
albumin
53
Young
Elde r ly
Flow rate

Graph.3. Comparing variables among young and elderly in Gingivitis patients
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Salivary total
protein
Young
Elde r ly
Salivay Albumin Flow Rate
Graph.4. Comparing variables among young and elderly in Periodontitis
patients
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Salivary total
protein
Young
Elde rly
Salivary albumin Flow rate
54

Graph.5. Comparing pH among young and elderly in different subgroups.
7
6
young
Elderly
Control Gingivitis Periodontitis
Graph.6. Comparing buffering capacity among young and elderly in different
subgroups.
7
6
5
Young
Elderly
Control Gingivitis Periodontitis
Graph.7.Gender variations among variables
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Salivary total
protein
Salivary
albumin
55
Males
Fem ales
Flow rate

6.DISCUSSION
Saliva is a complex substance that offers various opportunities to assess
general illness and to monitor disease and health status. The present study included
120 subjects, grouped as Young and Elderly. Further subgroups of 20 subjects each
were made as Controls, Gingivitis and Periodontitis subjects under each group.
Parameters like salivary total protein, salivary albumin, pH, buffer capacity and flow
rate were estimated to assess the role of these parameters as markers in periodontal
disease in order to place the diagnosis and management of the disease on a more
rational basis and to establish role of saliva as an indicator of mucosal integrity.
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 subgroup were 0.86 (S.D=0.21)g/ml, 1.19g/ml
(S.D=0.23) & 1.59 g/ml (S.D=0.48). The rise in these values were statistically very
highly significant (p=0.001).The study conducted by Henskens et al,( 1993) 33 was in
accordance with the same. It showed the mean values in the controls, gingivitis and
periodontitis subgroup as 1.06 (S.D=0.25)mg/ml, 1.49mg/ml (S.D=0.58) & 2.21
mg/ml (S.D=1.0). In the present study additional data was collected to compare the
parameter in the young and the elderly. Aged individuals did not seem to vary much
from the young in the secretion of salivary proteins. Even the rise in total protein
concentration in these groups was similar. Both the groups showed 1.8 and 1.3 times
value rise in periodontitis & gingivitis subgroup respectively, when compared to that
of the controls.
56

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’s and crevicular fluid proteins. Thus, the elevated protein levels are
most likely due to enhanced synthesis and secretion by the individual glandular
saliva’s. Hormia et al(1993) 128 reported higher concentration of epidermal growth
factor in glandular saliva’s in periodontitis. Also glandular derived proteins, Cystatin
C and amylase showed significant rise in periodontitis subjects proving the glandular
origin of these proteins (Henskens et al,1996) 106 .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.
Salivary albumin estimation was carried out in all the subgroups of both the
groups. 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 to the controls. The
groups showed around 4-5 and 2-3 times rise in periodontitis & gingivitis subgroup
respectively, when compared to that of the controls. Total mean salivary albumin for
controls, gingivitis and periodontitis patients was 0.09(S.D=0.04), 0.24(S.D=0.09),
and 0.44(S.D=0.12) mg/ml. The rise in these values were 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 GCF (Henskens et al,1993;Basu et al,1984) 33,129 . It showed the mean values in
the controls, gingivitis and periodontitis subgroup as 0.08 (S.D=0.05)mg/ml,
57

0.30mg/ml (S.D=0.30) & 0.67 mg/ml (S.D=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. The salivary albumin levels that were
found in subjects with gingivitis and periodontitis indicate that it is probably caused
by leakage of plasma proteins due to inflammation. Saliva from totally edentulous
patients contained five to six times less albumin than saliva from control, confirming
the sulcular origin for albumin (Terrapon et al,1996) 58 . This suggests the role of this
parameter as marker for gingivitis and periodontitis where plasma protein leakage
occurs as a consequence of the inflammatory process. Saliva can also be used as an
indicator of prognosis during periodontal treatment. Henskens et al (1996) 106
evaluated the effect of periodontal treatment on the protein composition of whole and
parotid saliva. Significant changes in salivary protein composition including that of
albumin occurred only in whole saliva, after treatment.
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 (Bratthall and Carlsson, 1986) 130 . It is in accordance with 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. One of the
previous study suggested that no differences were found in the flow rate of saliva
between the periodontitis group and the control groups (Sewon et al,1990) 75 .However
the mean flow rate in the healthy elderly was only 0.38 ml/min and it was a very
highly significant difference when compared among all the subgroups of the young
58

and elderly. 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 (Shern et al,1993;
Närhi et al,1999) 131, 93 . However, some studies suggest that unstimulated salivary
flow is related to age (Navazesh et al, 1992, Percival et al, 1994; Yeh et al,
1998) 81,94,132 . It has also been suggested that there are some age-related alterations in
salivary function (Yeh et al., 1998) 132 .
As shown in the present study there was a significantly decreased flow rate
in females when compared to males (p=0.001). This difference has been suggested to
be due to the size of the salivary glands (Dawes et al., 1987) 86 . Also in menopause,
many women seem to suffer from xerostomia, which then ameliorates in older age
(Parvinen and Larmas, 1982) 90 . In a study by Tarkkila et al. (2001) 133 it was found
that the occurrence of dry mouth in menopause-aged women was as high as 19.9% (n
= 631) 133 .
pH and buffer capacity estimation of the study showed that there was no
significant correlation between pH and buffering capacity in gingivitis and
periodontitis patients when compared with the controls both in young and the elderly.
However a interesting finding was significant rise in the buffering capacity among the
elderly controls when compared with the young. Galgut 78 in his study had tried to
correlate between pH and gingivitis & periodontal pockets. Statistically significant
correlations between gingivitis and pH did not exist, but significant correlations did
exist between pH and periodontal pockets 78 . The pH in the gingival crevice may vary
between 7.5 and 8.5. Placement of the pH paper in the above mentioned study in the
59

periodontal pocket rather than it’s estimation in the collected saliva sample as in
present case may be the reason for insignificant pH changes in our study. Watanabe et
al (1996) 79 had conducted a study which showed that pH values in the periodontal
pockets differed among the measuring points and were changeable. Also, the salivary
pH showed no correlation with that in the periodontal pockets (Watanabe et al ,1996)
79 which is in accordance with our findings. One of the study mentions that the
elevation of pH in the blood and saliva of the oral cavity to above 7.6 seen in their
study was responsible for growth of dental plaque crystals that caused periodontal
disease 74 .In another study conducted among periodontitis positive and negative
patients, no difference was found in the buffering capacity of saliva between the
groups (Sewon et al,1990) 75 . Our finding correlates with Sewon et al’s (1990) 75
study, 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.
60

7.CONCLUSION
In the oral cavity, proteins, especially albumin is considered as a serum
ultrafiltrate to the mouth .Gingivitis and periodontitis are oral diseases that are
characterised by chronic inflammation and loss of mucosal integrity. Thus these
diseases were considered in the present study .The study aimed at both the young and
the elderly groups comparing salivary total protein, albumin, pH, buffering capacity
and flow rate in different subgroups.
A very highly significant rise in the salivary total protein and albumin
concentration was noted in both young and elderly subjects of our study. This
suggests the role of these parameters as markers for gingivitis and periodontitis where
plasma protein leakage occurs as a consequence of the inflammatory process. These
parameters thus, can also be used as indicators of loss of mucosal integrity. Since
there were no changes in the protein rise among the young and elderly, it is evident
that old age as such need not affect the composition of saliva.
The present study showed that flow rates do not alter with periodontal status,
but alters significantly with age. Other parameters like salivary pH and buffering
capacity does not alter significantly with gingivitis and periodontitis .An overall
decrease in salivary flow rate is observed among the elderly in the present study. Also
salivary flow rate of women was significantly lower than that of men. However, a
longitudinal study would be required to draw definite conclusions.
61

SUMMARY- The present study included 120 subjects, who were
grouped based on their age as Young and Elderly. Further subgroups of 20 subjects
each were made as Controls, Gingivitis and Periodontitis subjects under each group.
Unstimulated saliva was collected from patients of all the subgroups in both the
groups. Flow rate was noted down during collection of the sample. Parameters like
salivary total protein, salivary albumin, pH, & buffer capacity were estimated to
assess their role as markers of periodontal disease. Salivary protein estimation was
done using Biuret method and salivary albumin was assessed using Bromocresol
green method. pHmeter was used to estimate pH & buffer capacity was estimated by
titration .The results were tabulated and analysed statistically by ‘ANOVA’ , students
‘t-test’ and Tukey HSD tests. A comparative analysis of all the parameters in the
subgroups of both the groups was done. Also the parameters were correlated among
the young and the elderly and between males and females.
Very significant rise in the salivary total protein and albumin was noted
in gingivitis and periodontitis patients when compared with the controls in both the
groups suggesting their role as markers for gingivitis and periodontitis. Mean values
of salivary total protein in the controls, gingivitis and periodontitis subgroup were
0.86 (S.D=0.21), 1.19(S.D=0.23) & 1.59 (S.D=0.48) mg/ml respectively. Mean values
of salivary albumin for controls, gingivitis and periodontitis patients was
0.09(S.D=0.04), 0.24(S.D=0.09), and 0.44(S.D=0.12) mg/ml respectively. Other
parameters like pH, buffering capacity and flow rate were not differing significantly
with the periodontal status in both the groups. An overall decrease in salivary flow
rate was observed among the elderly in the present study. Also salivary flow rate of
women was significantly lower than that of men.
62

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77

REGISTRATION NUMBER
ANNEXURE - I
CASE HISTORY PROFORMA
NAME AGE SEX
OCCUPATION
ADDRESS
DATE
CHIEF COMPLAINT.
HISTORY OF PRESENT ILLNESS
PAST HISTORY
H/O DIABETES DRUG ALLERGY
HYPERTENSION BLOOD TRANSFUSION
TUBERCULOSIS BLOOD DONATION
S T D OPERATION
PERSONAL HISTORY
HABITS DIET PAN CHEWING
FAMILY HISTORY
PHYSICAL EXAMINATION
SMOKING SLEEP
ALCOHOL
BUILT ANEMIA
PULSE RATE OEDEMA
BLOOD PRESSURE CLUBBING
RESPIRATORY RATE CYANOSIS
TEMPERATURE JAUNDICE
78

EXTRA ORAL EXAMINATION :
INTRA ORAL EXAMINATION :
PERIODONTAL STATUS :
TMJ LYMPHNODE STATUS
SOFT TISSUE HARD TISSUE
BUCCAL MUCOSA
LABIAL MUCOSA
PALATAL MUCOSA
GINGIVAL MUCOSA
LINGUAL MUCOSA
FLOOR OF THE MOUTH
ORAL HYGIENE : GOOD/ MODERATE/ POOR
GINGIVITIS – NIDR Gingival inflammation Index (Bleeding index)
0= No bleeding
1= Bleeding results after probe is placed in gingival sulcus upto 2 mm and
drawn along inner surface of the gingival sulcus.
PERIODONTITIS : LOCALIZED / GENERALIZED
POCKET DEPTH :
CALCULUS
18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28
48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38
79

SALIVA SAMPLE COLLECTION
FLOW RATE :
LABORATORY INVESTIGATIONS
SALIVARY TOTAL PROTEIN :
SALIVARY ALBUMIN :
SALIVARY pH :
BUFFER CAPACITY :
80

Method
Biuret method
Test principle
ANNEXURE 2
TOTAL PROTEIN ESTIMATION 127
Salivary protein forms a coloured complex with cupric ions in alkaline medium.
Requirements
1) Test.tubes : 15 x 125 mm
2 Graduated pipettes: 5 mI
3) Test-tube stand
4) Push button pipette of 0.05 ml
5) Colorimeter – autoanalyser.
Reagants used
Stock Biuret reagent: Dissolve 45 g of Rochelle salt in about 400 ml of 0.2 N
Sodium hydroxide and add 15 g of copper sulphate by stirring continuously until
the solution is complete. Add 5 g of potassium iodide and make upto a litre with 0.2
N sodium hydroxide.
1) Protein reagent (ready to use.): (Working Biuret reagent) – Reagent 1
Dilute 200 ml of stock reagent to a litre with 0.2 N sodium hydroxide which
contains 5g of potassium iodide per litre.
2) Protein standard: Reagent 2
6.0 g/dl: 6 g of bovine albumin dissolved in 100 ml of normal saline,
containing 0.1 g/dl, sodium azide.
81

3) Sample blank reagent: 9.0 g of Rochelle salt & 5 g of potassium iodide dissolved
in one litre of 0.2 N sodium hydroxide.
Stability of the reagents
Reagents 1 and 3 are stable at room temperature (25°C-15 o C) for one year. Reagent 2
(Protein standard) is stable at 2-8°C for one year.
Caution
Keep out of reach of children.
In case of contact with eyes, rinse with lots of water.
Wear suitable gloves and eye/face protection.
Always use safety pipettes to pipette out the reagents.
Keep the bottles tightly closed.
Sample preparation
Saliva sample can be stored upto 6 days at 4 o Celsius.
Procedure:
Required reagents were pipetted in clean and dry test tubes. One standard and one
blank were used for each assay series.
Table 15-Procedure
Sample
Reagent 1 1.00 ml
Sample 0.02 ml
Reagents were mixed as mentioned in table 15 and incubated at 20-30 o Celsius for 30
minutes. Absorbance of sample(A sample) against Reagent 1 ( reagent blank) was
read using a colorimeter. If readings could not be taken at 546 nm, the absorbance of
82

the standard was determined once for each assay series, using the standard provided
(Reagent 2), instead of sample material. The absorbance obtained (A standard) was
entered in the calculation.
Applicability of buiret method on autoanalysers (Colorimeter)
Wavelength : 546 nm(530-570 nm)
Cuvette : 1 cm light path.
Temperature: 20-30 degree Celsius.
Quality control
Use of clean glassware, accuracy of pipetting, proper temperature control etc, are the
factors that affect the results.
Calculation to arrive at final total protein concentration is :
Concentration of protein – C
C = 19 x A sample (g/100ml)
C = 190 x A sample (g/l)
C = 6 x a sample/A standard (g/100 ml)
C = 60 x A sample/ A standard (g/l)
Salivary total protein was expressed in mg/ml in the present study.
Details of the kit used
Capacity - 200 tests (2x100 ml)
Batch No- Cat.No.400294012.
Manufacturer-Nicholas Piramal India Limited, Navi Mumbai-400705
83

Principle
ANNEXURE 3
SALIVARY ALBUMIN ESTIMATION 55
The reaction between albumin in saliva and the dye Bromocresol-green produces a
change in colour that is proportional to the albumin concentration.
Requirements
1) Test.tubes : 15 x 125 mm
2) Graduated pipettes :5 ml
3) Test-tube stand
4) Push button pipette of 0.05 ml
5) Colorimeter – Autoanalyser
Reagent Composition
1) Reagent
It is prepared by mixing following chemicals in 900 ml of distilled water,
a) Succinic acid: 8.85 g.
b) Bromcresol green: 108 mg
c) Sodium azide: 100 mg
d) Brij-35: 4.0 ml
pH of this solution is adjusted by using 1N sodium hydroxide to 4.1. Final
volume is made to one litre by using distilled water.
2) Albumin standard 3.0 g/dl : Bovine albumin 3.0 g in 100ml of normal saline
containing 0.1 g/dl sodium azide.
3) Sample blank reagent: It contains 0.885 g of succinic acid, 10 mg of sodium
84

azide & 0.4 ml of Brij-35. pH of this solution is adjusted to 4.1
Stability of the reagents
Reagents 1 and 3 are stable at room temperature for (25 0 C-15°C) one year. Reagent
2 (Protein standard) is stable at 2-8°C for one year.
Caution
Keep out of reach of children.
In case of contact with eyes, rinse with lots of water.
Wear suitable gloves and eye/face protection.
Always use safety pipettes to pipette out the reagents.
Keep the bottles tightly closed.
Avoid direct exposure of working reagent to light.
Asay procedure:
Table 16- Laboratory Procedure
Blank
Standard Sample
Reagent 1.0 ml 1.0 ml 1.0 ml
Standard - 0.01 ml -
Sample - - 0.01ml
85

The agents were pipetted in clean and dry test tubes. They were mixed as mentioned
in table 16 and measured against reagent blank. Measurement was done with the help
of a colorimeter –autoanalyser using a red filter (630 nm).
Applicability of bromocresol green method on autoanalysers
Wavelength - 630 nm
Temperature - 37 o Celsius
Standard concentration - 3g/dl
Blank - Reagent
Reaction time - 1 min
Sample Volume - 10 microlitre
Reagent Volume - 1000 microlitre
Cuvette - 1 cm light path
Zero setting with : reagent blank
Final Calculation to get the albumin level is
Albumin g/ml= A (Sample- Reagent blank)/ A (Standard- Reagent blank) x 3 g/dl
Salivary albumin was expressed in mg/ml in the present study.
Details of the kit used
Albumin colorimetric test
Capacity - 5x 50 ml
Batch No- - AFP 11000
Manufacturer- Agappe diagnostics,Thane- 401210
86

ANNEXURE 4
pH AND BUFFER CAPACITY 69
The most reliable and convenient method for measuring pH is by the use of a
pH meter
Principle
When the pair of electrodes or a combined electrode (glass electrode and calomel
electrode) is dipped in an aqueous solution, a potential is developed across the thin
glass of the bulb (of glass electrode). The e.m.f. of complete cell (E) formed by the
linking of these two electrodes at a given solution temperature is therefore:
Eref which is the potential of the stable calomel electrode at normal room temperature
is + 0.250V.
Eg1ass which is the potential of the glass electrode which depends on the pH of the
solution under test.
The resultant small e.m.f can be recorded potentiometrically by using vacuum tube
amplifier. Variations of pH with E may be recorded directly on the potentiometer
scale graduated to read pH directly.
Important Components of a pH Meter
1) Glass electrode
It consists of a very thin bulb about 0.1 mm thick blown on to a hard glass tube of
high resistance. The bulb contains 0.1 mol/litre HCI connected to a platinum wire via
a silver-silver chloride combination.
2) Calomel electrode
It consists of a glass tube containing saturated KCI connected to platinum wires
through mercury- mercurous chloride paste.
87

Determination of pH
1) The pH meter was turned on for 15 minutes and temperature knob was kept at
room temperature.
2) The read button was adjusted at STD (standard) position.
3) The scale pointer of the pHmeter was adjusted to zero.
4) The electrodes were immersed in standard buffer of pH 4.Then the range
switch was adjusted at pH 0-7.Read switch was placed at read position. If the
reading was not at 4, it was adjusted at 4 using calibration knob.Then, read
switch was again adjusted at STD position.
5) Electrodes were washed in a jet of distilled water and were cleaned with a soft
tissue paper.
6) Another standard buffer of pH 7 was used to standardize the pH meter.
7) Then, the electrodes were placed in salivary sample and read button flipped to
read position. pH reading was then taken down.
BUFFERING CAPACITY ESTIMATION 69
Buffering capacity can be defined as number of equivalent strong alkali or strong acid
required to be added to a liter of the buffer solution so as to change it’s pH by one.
Aim – To determine the buffering capacity of the salivary sample.
Principle – The sample whose pH is known is first titrated against standard sodium
hydroxide till the pH is raised by 1 unit, and then it is titrated against standard
hydrochloric acid till the pH is lowered by 1 unit. Buffering capacity is then
calculated.
88

Reagents
1) Phenol Red indicator
0.1 gm of phenol red in 5.7 ml of N/20 sodium hydroxide diluted to 250 ml with
distilled water.
2) Methyl red indicator
0.05 gm of methyl red dissolved in 100 ml of 50% alcohol.
3) 0.1 N Sodium hydroxide-standardized against oxalic acid.
4) 0.1 N Hydrochloric acid - standardized against standard sodium hydroxide.
Procedure
1 ml 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 colour 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 1 unit.
The titre values were noted down. The buffering capacity of the saliva towards acidic
and alkaline side was then calculated.
Calculations
Buffering capacity = Titre value x Normality x 1000
__________________________
1000 x 10
Buffering capacity was calculated using the above formula against standard sodium
hydroxide & standard hydrochloric acid .They were added up and the final value was
expressed in pH units by deducting the buffering capacity value from the pH of the
salivary sample.
89

ANNEXURE 5
The values of the parameters ,that are salivary total protein, salivary albumin,
pH, buffer capacity, and flow rate of our study sample are tabulated in tables 1-6.
Table 1 Group: Young - Subgroup : Control
No. Sex pH Buffer
Capacity
Total
protein
mg/ml
Albumin
mg/ml
1 F 6.5 5.2 0.5 0.2 0.3
2 F 6.0 5.2 0.6 0.2 0.5
3 F 6.7 5.5 0.5 0.05 0.5
4 F 6.5 6.1 1.0 0.08 0.6
5 M 6.5 6.1 0.9 0.1 0.7
6 F 7.0 6.2 1.0 0.05 0.5
7 F 7.5 6.3 1.0 0.2 0.4
8 M 7.2 6.1 0.9 0.1 0.8
9 F 5.8 5.6 0.6 0.1 0.5
10 F 6.5 5.8 0.9 0.2 0.4
11 F 6.0 5.5 1.1 0.08 0.5
12 M 6.0 5.5 1.2 0.07 0.7
13 F 6.5 5.8 0.9 0.1 0.5
14 M 6.5 6.0 1.0 0.08 0.7
15 M 6.7 6.2 0.9 0.05 0.6
16 F 6.0 5.8 1.0 0.1 0.4
17 F 6.5 5.5 0.9 0.06 0.4
18 M 7.0 6.0 1.1 0.07 0.5
19 M 7.2 6.1 0.8 0.05 0.6
20 M 7.0 5.8 1.1 0.1 0.7
90
Flow
rate
ml/min

Table 2 Group: Young - Subgroup : Gingivitis
No. Sex pH Buffer
Capacity
S.Total
protein
mg/ml
S.Albumin
mg/ml
1 F 6.5 6.1 1.3 0.3 0.4
2 M 6.5 5.3 1.2 0.3 0.6
3 M 6.5 6.1 1.1 0.2 0.5
4 M 6.4 6.1 1.3 0.3 0.6
5 M 7.0 6.2 1.2 0.2 0.7
6 F 6.2 5.8 2.1 0.2 0.5
7 M 6.8 6.0 1.9 0.3 0.8
8 M 6.4 5.8 1.0 0.3 0.6
9 M 6.2 5.2 1.0 0.4 0.7
10 F 6.4 5.9 1.2 0.3 0.4
11 M 7.0 5.8 1.1 0.2 0.6
12 M 7.0 5.2 1.0 0.1 0.6
13 F 7.2 6.8 1.0 0.3 0.4
14 M 7.1 6.1 1.1 0.2 0.5
15 M 6.6 5.5 1.4 0.1 0.6
16 F 6.8 5.8 1.0 0.3 0.4
17 F 6.2 6.1 1.1 0.2 0.3
18 F 6.6 6.0 1.2 0.1 0.4
19 F 5.9 4.9 1.4 0.3 0.4
20 M 6.5 5.8 1.2 0.2 0.5
91
Flow rate
ml/min

Table 3 Group: Young - Subgroup : Periodontitis
No. Sex pH Buffer
Capacity
S.Total
protein
mg/ml
S.Albumin
mg/ml
1 F 7.0 6.2 1.6 0.4 0.4
2 M 7.0 6.2 1.4 0.3 0.7
3 M 7.5 6.3 1.1 0.4 0.6
4 M 7.0 6.2 0.7 0.5 0.6
5 F 6.6 5.8 1.6 0.3 0.5
6 M 6.2 5.8 2.0 0.4 0.4
7 M 6.7 6.2 1.3 0.2 0.6
8 F 6.4 6.1 1.2 0.6 0.4
9 F 6.8 6.2 2.0 0.5 0.3
10 F 6.4 5.8 2.2 0.4 0.6
11 F 6.8 6.2 1.0 0.3 0.4
12 M 6.5 5.8 2.2 0.5 0.8
13 M 7.0 6.4 2.0 0.6 0.7
14 F 7.0 6.4 1.8 0.4 0.3
15 F 6.0 5.5 1.5 0.6 0.4
16 F 6.0 5.5 2.1 0.4 0.5
17 M 7.0 6.4 2.0 0.5 0.5
18 F 6.5 5.8 1.7 0.3 0.4
19 M 7.0 5.8 1.8 0.4 0.6
20 F 6.2 6.1 1.3 0.3 0.4
92
Flow
rate
ml/min

Table 4 Group: Elderly - Subgroup: Control
No.
Sex pH Buffer
Capacity
S.Total
protein
mg/ml
S.Albumin
mg/ml
1 M 6.5 6.2 1.1 0.1 0.4
2 F 6.5 6.4 0.6 0.09 0.3
3 M 6.5 5.5 0.8 0.08 0.5
4 M 7.0 5.8 0.5 0.1 0.4
5 F 6.6 6.0 0.7 0.1 0.3
6 F 6.5 6.1 1.0 0.08 0.2
7 F 7.0 5.8 1.1 0.07 0.3
8 M 7.2 5.8 0.6 0.09 0.5
9 F 7.0 6.0 0.4 0.06 0.4
10 M 6.5 5.8 0.7 0.1 0.3
11 M 6.0 5.5 1.1 0.1 0.5
12 M 7.2 6.1 0.8 0.06 0.6
13 F 6.0 5.8 0.5 0.07 0.3
14 F 6.5 5.8 1.0 0.07 0.2
15 M 7.0 6.0 0.8 0.09 0.5
16 F 7.2 6.2 1.2 0.1 0.3
17 M 6.5 6.1 0.9 0.08 0.6
18 M 6.6 6.2 1.0 0.09 0.4
19 F 7.0 6.0 0.8 0.1 0.5
20 F 7.5 7.0 1.1 0.1 0.4
93
Flow
rate
ml/min

Table 5 Group: Elderly - Subgroup : Gingivitis
No. Sex pH Buffer
Capacity
S.Total
protein
mg/ml
S.Albumin
mg/ml
1 M 6.0 5.5 1.0 0.4 0.5
2 M 6.5 5.8 1.3 0.3 0.4
3 M 6.0 5.9 1.4 0.2 0.3
4 F 6.5 5.4 1.1 0.3 0.1
5 F 6.0 5.5 1.0 0.4 0.2
6 M 6.5 6.1 1.2 0.3 0.4
7 M 6.7 6.1 0.9 0.3 0.4
8 F 6.7 6.0 1.1 0.1 0.3
9 F 7.0 5.8 1.3 0.2 0.2
10 M 7.2 5.7 1.0 0.3 0.5
11 M 7.5 6.6 1.4 0.4 0.4
12 F 7.2 6.1 1.1 0.2 0.3
13 M 7.0 5.5 1.2 0.1 0.6
14 M 6.7 6.1 1.0 0.2 0.4
15 M 6.5 6.0 1.1 0.3 0.5
16 F 7.0 5.2 1.3 0.3 0.5
17 F 6.5 5.8 1.3 0.2 0.3
18 F 6.7 5.5 1.2 0.1 0.4
19 F 6.5 5.6 0.9 0.4 0.3
20 F 6.7 5.4 1.0 0.1 0.2
94
Flow rate
ml/min

Table 6 Group: Elderly - Subgroup : Periodontitis
No. Sex pH Buffer
Capacit
y
S.Total
protein
mg/ml
S.Albumin
mg/ml
1 M 6.7 6.1 1.0 0.3 0.4
2 F 6.5 5.9 1.3 0.4 0.3
3 F 6.5 6.1 1.8 0.3 0.2
4 M 7.0 6.2 1.5 0.6 0.5
5 M 6.5 5.8 0.8 0.3 0.5
6 M 7.5 6.9 1.4 0.4 0.4
7 M 6.5 6.0 1.1 0.5 0.4
8 M 6.5 6.0 2.1 0.5 0.3
9 M 6.5 6.0 1.7 0.4 0.4
10 M 6.5 6.3 0.9 0.6 0.5
11 F 6.5 5.8 2.3 0.3 0.2
12 F 6.5 6.0 2.2 0.4 0.3
13 M 6.5 6.0 2.1 0.7 0.4
14 F 6.5 6.0 0.6 0.5 0.2
15 F 6.5 5.8 0.9 0.3 0.1
16 M 6.7 6.5 2.2 0.5 0.3
17 M 6.5 6.1 2.3 0.6 0.4
18 F 6.5 6.1 1.5 0.4 0.1
19 F 6.5 6.4 1.6 0.6 0.2
20 M 7.0 6.2 1.8 0.7 0.3
95
Flow
rate
ml/min