Scope of saliva as a diagnostic fluid/prosthodontic courses

Scope of saliva as a diagnostic
fluid
INDIAN DENTAL ACADEMY
Leader in continuing Dental Education
www.indiandentalacademy.com

contents
• Introduction
• Composition of saliva
• Collection of saliva
• Saliva as diagnostic fluid
• conclusion

• “Saliva is the clear mucoserous
secretion formed mainly in salivary
glands which has lubricative,
cleansing & anti microbial, excretory
and digestive functions”.

Based on their anatomical size
 MAJOR SALIVARY GLANDS
Parotid gland 30%
Submandibular gland 60%
Sublingual gland 5%
 MINOR SALIVARY GLANDS 1%
Buccal
Labial
Lingual
Palatine
Glossopalatine

BASED ON HISTO CHEMICAL NATURE OF SECRETION
SEROUS, MUCOUS, MIXED
 Parotid glands  Purely serous
 SubmandPredominantlyserousMixed
 Sublingual  Pre. mucousMixed
 Labial,Buccal Pre.mucousMixed
 Palatine, GlossopalatinePurely mucous
 Anterior lingual glands  chiefly mucous
 Posterior part of the tongue mixed
 Von Ebner’s GlandsPurely serous

COMPOSITION OF SALIVA

“ ORGANIC COMPONENTS
1)Salivary proteins : Mucins, statherins, histatins,
immunoglobulin etc.
2) Digestive enzymes: α – amylase, lipase, peroxidase,
lactoferrins, cystatins.
3) Carbonic anhydrase
4) Intrinsic growth factors: EGF , TGF α β
5) Circulating adrenal glucocorticoids: Coritsol.
6) Sex hormones in blood: Estriol.
7) Blood glucose in lower levels.
8) Blood group substances from A B O groups.

INORGANIC COMPONENTS
Mainly
•Sodium (Na +),
•Potassium (K +)
•Hydroxyappitite & calcium phosphate salts.
•Chloride (Cl -) , Carbonic acid (Hco 3 - )
Smaller amounts
•Magnesium (Mg 2+)
•Thiocynate (SCN -)
•Fluoride and Iodine

Saliva also contains constituents that do not
originate in the salivary gland
• including gingival crevicular fluid
• serum transduate from the mucosa and
sites of inflammation
• epithelial and immune cells
• microorganisms.

Factors affecting salivary composition

Normal salivary flow
• In general, healthy adults produce 500–1500
mL of saliva per day, at a rate of approximately
0.5 mL/min
• USFR < 0.1 mL/min
• SFR < 0.7 Ml/min
• are considered abnormally low, indicative of
salivary gland hypofunction.

• When a person is in a resting state, saliva
production is largely produced by the
submandibular gland ( 2/3rd )
• In contrast, when saliva production is
stimulated either via chewing gum or plastic
(e.g. parafilm), or through acid stimulation,
most of the saliva produced is from the
parotid gland( ½)

Factors affecting salivary flow
• Patient position
• Hydration
• Diurnal
• Seasonal variation

TYPES OF SALIVA
• Gland specific : The collection and evaluation of the secretions from
the individual salivary glands
• primarily useful for the detection of gland-specific pathology, i.e.,
infection and obstruction.
• Whole saliva: mixture of oral fluids ,includes
• secretions from both the major & minor salivary glands
• several constituents of non-salivary origin such as (GCF)
• expectorated bronchial and nasal secretions,
• serum and blood derivatives from oral wounds,
• bacteria & their products, viruses, fungi,
• desquamated epithelial cells, cellular components, food debriswww.indiandentalacademy.com

METHODS OF COLLECTION OF SALIVA

Precautions to be taken before
sample collectionAvoid
• alcohol for 12 hrs & dairy products for 20 min
• foods with high sugar or acidity, or high caffeine
content before sample collection. (may lower ph or
increase bacterial growth)
• Do not eat a major meal within 60 minutes
before sample collection.
• Document consumption of alcohol, caffeine, nicotine, &
any medication usage.www.indiandentalacademy.com

• Rinse mouth with water to remove food
residue before sample collection, and swallow
to increase hydration.
• Wait at least 10 minutes after rinsing before
collecting saliva to avoid sample dilution.
• It is also advisable to document the physical
activity level of research participants and the
presence of oral diseases.

BLOOD CONTAMINATION OF SALIVA
• Even an invisible amount of blood contamination has the
potential to falsely elevate salivary analyte levels
• participants should not brush their teeth within 45 minutes
• Dental treatment should not be performed within 48 hours
prior to sample collection.
• Participants should be screened for oral health problems
or injuries.
• Saliva samples visibly contaminated with blood should be
discarded and recollected.www.indiandentalacademy.com

• METHODS FOR COLLECTING WHOLE SALIVA
• Draining
• Suction
• Spitting
• Absorbent (sponge) method

SPITTING METHOD
• In spitting method pt allows saliva to accumulate in
mouth and then expectorates into a preweighed
tube usually once every 60sec for 5 to 15 min
ABSORBENT METHOD
• In absorbent method a pre weiged gauge sponge is
placed in pts mouth for a set amount of time.

• If stimulated whole saliva collection is required
• Standardised method of stimulation should be used
• Chewing unflavoured gum base
• Inert material like paraffin
• Rubber band
• At a controlled rate usually 60 tyms/min is reliable
& reproducible means
• 2% citric acid can also be placed on tongue at 30 sec
interval.

Lashley cup/ Carlson – Crittenden
Collector
• Used for parotid
• designed by carlson -
crittenden
• 2 metal cups with 2 outlet
tubes
• Inner cup collects saliva
• Outer cup connected to
vacuum
• Basic design was modified by
lashley

• Modified carlson-crittenden collector/ lashleys
cannula
• Single cup with two concentric chambers
• The inner chamber can be positioned over parotid
duct orifice for collection of saliva
• While suction applied to outer chamber holds cup in
place.

• Parotid saliva can be stimulated by applying 1ml of
2% citric acid to dorsum of tongue every 30 sec
• Depending on age & health status of subject flow
rate is usually 1 to 3 ml/min

Schneyers apparatus

Segregator

SCOPE OF SALIVA AS DIAGNOSTIC FLUID
Though saliva lacks
Drama of blood
Sincerity of sweat
Emotional component of tears
It is a unique fluid Which harbors a wide spectrum of
Proteins, nucleic acids, electrolytes, and hormones that
originate from multiple local and systemic sources.
saliva reflects the body’s health and well-being

• Salivary diagnosis is an increasingly important
field in
• Dentistry
• Physiology
• Internal Medicine,
• Endocrinology
• Pediatrics
• Immunology
• Clinical Pathology,
• Forensic Medicine,
• Psychology
• Sports Medicine

ADVANTAGES OF SALIVA ( WHEN COMPARED TO
SERUM ) AS DIAGNOSTIC FLUID
• collected noninvasively by individuals with modest
training.
• Easy to store & transport
• Cost effective approach for the screening of large
populations.
• Multiple collections can be performed without
imposing too much discomfort on the donor

• Gland-specific saliva can also be used for diagnosis
of pathology specific to one of the major salivary
glands.
• Minimal risk of contracting infections during saliva
collection
• saliva can be used in clinically challenging
situations, such as obtaining samples from
children , handicapped or anxious patients in whom
blood sampling could be a difficult act to perform.

DISADVANTAGES OF SALIVA AS DIAGNOSTIC
TOOL
• Diurnal /circadian variations of certain biomolecules
present in saliva
• It does not always reliably reflect the concentrations
of these molecules in serum.
Salivary composition can also be influenced by
• method of collection
• Degree of stimulation of salivary flow

• Saliva contains analytes in concentrations that are
1000-fold less than those in blood.
• Sensitive detection systems are needed.

• Most of the biomolecules that are present in saliva
are either synthesized in situ in the salivary glands
and/or transported from blood capillaries into
saliva
• understanding of the biomolecules present in saliva
during a normal healthy physiological state, as
opposed to a pathological condition, required in
order for saliva to become a sample of choice for
diagnostic and treatment purposes.

Transfer of biomolecules from blood
to saliva
DIFFUSION
ACTIVE
TRANSPORT
ULTRA
FILTRATION
TRANSUDATION

DIFFUSION
• common route for substances to migrate from blood to
saliva is via unaided or passive diffusion
• capillaries surrounding the glands are quite porous for
many small molecules.
• A serum molecule must cross 5 barriers to reach saliva :
1. The Capillary Wall
2. The Interstitial Space;
3. The Basal Cell Membrane Of The Acinus Cell / Duct Cell;
4. The Cytoplasm Of The Acinus Or Duct Cell
5. The Luminal Cell Membranewww.indiandentalacademy.com

• Ability of molecule to pass depends on its size, and
electrical charge
• If a molecule is polar in nature, & separates into
charged ions while in solution, it is difficult to pass
• steroid hormones are relatively small in size, &
composed of fatty acids, so they tend to pass relatively
easily by diffusion.
• Molecules that are bound to large carrier proteins,
such as serum albumin, are too big to enter by this
route

ACTIVE TRANSPORT
• route used by secretory IgA (SIgA).
• polymeric IgA, which is secreted by B-lymphocyte
cells in close proximity to salivary cells, is then
bound by IgA receptors present on acinus cells, &
then gets released into saliva

Lumen

Ultrafiltration
• Filtration through the spaces between acinus and
ductal cells.
• Also through gap junctions between cells of secretory
units ( intracellular nexus)
• To follow this type of transportation into saliva, the
molecules must be relatively small.( < 1900da)
• such as water, ions, catecholamines, and steroids
• Sulfated steroids, estroil steroids cannot pass through pl
bilayer due to their charge

Transudation:
• Transudation of plasma components into oral
cavity either from crevicular fluid/ directly
from mucosa
• Plasma albumin in saliva mostly occurs
through this.

• Diagnosis of oral diseases
• Caries activity tests
• Systemic diseases
Hereditary
Autoimmune
Malignancy
Infectious
Cvs, renal
• Drug monitoring
• Monitoring level of hormones
• Forensic odontology

Diagnosis of Oral Disease
• Quantitative alterations in saliva may be a result of
medications. At least 400 drugs may induce xerostomia.
• Diuretics, antihypertensives, antipsychotics,
antihistamines, antidepressants, anticholinergics,
antineoplastics, and recreational drugs such as opiates,
amphetamines, barbiturates, hallucinogens, cannabis,
and alcohol have been associated with a reduction in
salivary flow

• Reduced salivary flow may lead to oral problems like
progressive dental caries, fungal infection, oral pain,
and dysphagia.
• Saliva can be used for the detection of oral
candidiasis, and salivary fungal counts may reflect
mucosal colonization.

Dental caries
• Saliva may also be used for the monitoring of oral
bacteria.
• Anaerobic bacteria can survive in saliva, and can utilize it
as a growth medium
• Studies have shown that salivary parameters such as flow
rate, viscosity, ph, buffering capacity were decreased in
subjects with high risk of caries
• So saliva testing is an important part in routine protocol
while diagnosing & treating dc.

Caries activity tests
Lactobacillus colony count test
• Introduced by HADLEY in 1933
• Estimates no of acidogenic bacteria
• Saliva is collected by chewing paraffin wax for 3min (
before breakfast)
• specimen is vigourously Shaken for 2 minutes.
• Diluted with Distilled Water
• Diluted sample are spread evenly on Petri dishes
• Containing 20ml of cooled Liquefied Agar
( Rogasa’s SL agar plate )

• The plates are incubated for 3to4 days at 37C
• Number of Lactobacillus colonies are counted
No of
organisms per
cc
Symbolic
designation
Degree of
caries activity
suggested
0 – 1000
1000- 5000
+/-
+
Little or None
Slight
5000- 10,000 ++ Moderate
More than
10,000
+++or++++ Marked

CALORIMETRIC SNYDER TEST
• The ability of salivary microorganism to form organic acids from a
carbohydrate medium
• The glucose agar medium contains an indicator dye, Bromocresol
green.
• Changes color from green to yellow in the range of pH 5.4 to 3.8
• Salivary sample is collected & 0.2 cc of saliva is pipetted into the
melted medium at 50c
• The incubation period is up to 72 h at 37c
• The rate of color change from green to yellow is indicative of the
degree of caries activity. www.indiandentalacademy.com

24 hours
If yellow
48 hours
If yellow
72 hours
If yellow
Marked caries
susceptibility
Definite caries
susceptibility
Limited caries
susceptibility
If Green
Continue to
incubate &
observe at 48 h
If Green
Continue to
incubate &
observe at 72 h
If Green
Caries inactive

Streptococcus mutans level in Saliva
• Measures the no of streptococcus mutans CFU
per unit volume of saliva.
Procedure
• Sample of organisms is obtained by the use of
tongue blades ( Wooden spatulas)
• Then pressed against Streptococcus mutans selective
MSB ( Mitus salivarius Bacitracin ) Agar in special
Petri dishes.

Interpretation
• Level of Streptococcus Mutans>10 /ml of saliva
= Unacceptable.
Disadvantages
• Difficulty of Distinguishing b/w a carrier state and
cariogenic state
• SM may constitute less than 1% of total flora of
plaque
• SM tends to be located at specific site only.

ENAMEL SOLUBILITY TEST
• When Glucose is added to the saliva containing
powdered Enamel, Organic acids are formed.
• this acids Decalcify the Enamel
• Resulting in an increase in the amount of soluble
calcium in the saliva – glucose – enamel mixture.
• Extent of increase in calcium is a direct measure of
the degree of caries susceptibility.

SALIVARY REDUCTASE TEST
• Measure the activity of the reductase enzyme
present in salivary bacteria.
• Kit – Treatex.
• Saliva is collected in a plastic container.
• Sample is then mixed with the dye
Diazoresocinol
• Color changes and the caries conduciveness
• Reading is taken after 15 minutes
• No incubation procedure

ALBAN TEST
Materials
• Snyder test agar
• A small scale to measure 60 grams
• A 2 liter Pyrex glass to melt the medium
• A funnel to dispense the medium into test
tubes
• 100mm,16mm test tubes with screw caps.

Procedure
• 60 grams of Snyder test agar is placed in 1 liter of
water
• The suspension is brought to a boil over a low flame.
• Melted agar is distributed using about 5ml/tube
• Autoclaved for 15 min
• Allowed to cool
• Stored in a refrigerator

• 2 tubes of alban medium are taken from the
refrigerator.
• Pt is asked to expectorate a small amount of
saliva directly into the tubes
• Labeled
• Incubated at 98.6F for up to 4 days
• The tubes are observed daily

Scale for Scoring
• No color change
• Beginning Color change = ‘ + ’
( from top of medium down )
• One half color change = ++
• ¾ color change = +++
• Total color change = ++++
From bluish green to definite yellow
pH 5 to 4

• Reading -ve - Labelled Negative
• All other readings - +ve
• Slower change is - Improved
• Faster change - Worse
• Consecutive readings
are nearly identical – No change

Periodontitis
• Specific immunoglobulins in saliva directed toward
periodontal pathogens have been identified.
• saliva from treated periodontitis patients had higher IgA
and IgG levels to periodontalpathogens porphyromonas.
gingivalis and Treponema. denticola than as compared
to saliva from control subjects
• Patients with periodontal disease have demonstrated
high levels of this peroxidase enzyme in saliva

• Elevated Ca concentration in saliva
• Volatile sulfur compounds, primarily hydrogen sulfide
and methylmercaptan, are associated with oral malodor.
• Salivary volatiles have been suggested as possible
diagnostic markers and contributory factors in
periodontal disease.
• . Circulating C-reactive protein may reach saliva via GCF
or the salivary glands. High levels of C-reactive protein
have been associated with chronic and aggressive
periodontal diseases

Recurrent aphthous stomatitis :
• Evidence shows that there is increased oxidative stress
• levels of both plasma and salivary antioxidants are
decreased in patients with RAU.

Systemic diseases
Hereditary
Autoimmune
Malignancy
infectious

Cystic fibrosis (CF) :
• lethal, congenital, genetic disease of unknown
etiology.
• Defective regulation of ion and water transport in
exocrine glands and possibly also in other epithelial
cells has a central role in the pathogenesis of this
disease.
• Abnormally high calcium levels in some secreted
fluids and some cells of CF patients.

• Organs most commonly affected are
• Sweat glands – produce inc conc of nacl
• Pancreas - pancreatic insufficiency
• Lungs – develops copd
• Saliva of cf pts contain inc ca levels
• A factor produced by CF fibroblasts in culture can
increase the calcium concentration in healthy cells,
although this may be an indirect effect

• Inc in calcium protein aggregates in sub mand saliva
is seen causing turbidity
• Saliva contains an un usual epidermal growth factor
• Inc salivary pge2 is detected
• Parotid saliva donot demonstrate characteristic
changes

Sjogrens syndrome
• There was a reduced flow rate ( <0.1 ml/min)
• whole saliva flow rate (Sreebny and Zhu, 1996) and
gland-specific sialometry and sialochemistry (Kalk et
al., 2002) could be used to provisionally diagnose SS
• Measuring submandibular/sublingual flow rates may
contribute to an early diagnosis of ss.

Sialochemistry reveals
• The Na+ conc in the parotid glandular saliva is six fold
higher in SS patients as compared to non-SS
• Reduced phosphate level compared to normal
controls.
• Increase in IgA, IgG and lactoferrin.
• Albumin concentration was also elevated
• Inc beta 2 microglobulin
• Inc conc of cystatin c & s

Renal disease
• In patients with CRF increase in saliva urea, creatinine is
found.
• colormetric test strips were used to monitor
salivary nitrate and uric acid before and after
hemodialysis.
• It was suggested that a salivary test could be
used by patients to decide when dialysis is
required, thereby eliminating unnecessary visits
to a dialysis clinic

• Salivary phosphate has been successfully used as a
clinical biomarker forhyperphosphatemia, which is an
important contributor to cardiovascular calcification in
chronic renal failure (CRF)
• Evaluation of phosphate levels in saliva correlated
positively with serum creatinine & gfr . Thus, salivary
phosphate may provide a better marker than serum
phosphate for the initiation of treatment of
hyperphosphatemia in CRF and HD.

cvs
• C-reactive protein (CRP) can be monitored in salivary
samples,
• but CRP remains a non-specific inflammatory response
factor that increases in many conditions including
periodontal disease
• markers include CRP, myoglobin & myeloperoxidase, in
combination with an ECG, showed a highly significant
correlation with myocardial infarct patients as compared to
healthy controls.
• Robust clinical studies are required to validate salivary
biomarkers for CVD www.indiandentalacademy.com

Infectious diseases
Bacterial viral fungal protozoal
spirochetal

• Helicobacter pylori infection is associated with peptic
ulcer disease and chronic gastritis.
• Infection with this bacterium stimulates
the production of specific IgG antibody.
• An ELISA test for the detection of IgG antibody in
serum produced 97% sensitivity and 94% specificity
in detection of the disease
• sensitivity of saliva samples tested for the presence
of H. pylori DNA by (PCR) assay is 84%

• results also indicated that H. pylori exists in
higher prevalence in saliva than in feces, and the
oral-oral route may be an important means of
transmission of this infection in developed countries

• Evaluation of the secretory immune response in the
saliva of children infected with Shigella revealed higher
titers of anti lipopolysaccharide and anti-Shiga toxin
antibody in comparison with healthy controls.
• It was suggested that salivary levels of these
immunoglobulins could be used for monitoring of the
immune response in shigellosis

• Lyme disease is caused by the spirochete Borrelia
burgdorferi and is transmitted to humans by blood-
feeding ticks.
• The detection of anti-tick antibody in saliva has
potential as a biologic marker of exposure to tick
bites,
• This serve as a screening mechanism for individuals
at risk for Lyme disease

• Antibodies against viruses and viral components can be
detected in saliva and can aid in the diagnosis of acute
viral infections, congenital infections, and reactivation
of infection
• Saliva was found to be a useful alternative to serum for
the diagnosis of viral hepatitis. Acute hepatitis A (HAV)
and hepatitis B (HBV) were diagnosed based on the
presence of IgM antibodies in saliva

• Analysis of oral fluid samples collected with Orasure®
provided an excellent method for the diagnosis of
viral hepatitis B and C.
• Sensitivity and specificity of 100% for the detection
of antibodies for both diseases in oral fluid in
comparison with serum antibodies were reported

• Saliva may also be used for determining
immunization ,detecting infection with measles,
mumps, and rubella
• detection of antibodies in oral fluid samples
produced sensitivity and specificity of 97% and 100%
for measles,
• 94% and 94% for mumps
• 98% and 98% for rubella, respectively,
• In comparison with detection of serum antibodies for
these viruses www.indiandentalacademy.com

• The shedding of herpesviruses (human herpesvirus –8,
cytomegalovirus, and Epstein-Barr virus) in nasal secretions
and saliva of infected patients has been reported
• Other investigators suggested that reactivation of herpes
simplex virus type-1 (HSV-1) is involved in the pathogenesis of
Bell's palsy and reported that PCR-based identification of virus
in saliva is a useful method for the early detection of HSV-1
reactivation in patients with Bell's palsy.
• The shed HSV-1 virus was detected in 50% of patients with
Bell's palsy in comparison with 19% in healthy controls

• Dengue is a mosquito-transmitted viral disease.
• Primary infection of the virus may lead to a self-limiting
febrile disease
• secondary infection may cause serious complications like
dengue hemorrhagic fever or dengue shock syndrome.
• Salivary levels of anti-dengue IgM and IgG demonstrated
sensitivity of 92% and specificity of 100% in the diagnosis
of primary and secondary infection, and salivary levels of
• IgG proved useful in differentiating between primary and
secondary www.indiandentalacademy.com

• Studies have demonstrated that the diagnosis of infection
with the human immunodeficiency virus (HIV) based on
specific antibody in saliva is equivalent to serum in accuracy,
and therefore applicable for both clinical use and
epidemiological surveillance
• Antibody to HIV in whole saliva of infected individuals,
which was detected by ELISA and Western blot assay,
correlated with serum antibody levels
• As compared with serum, the sensitivity and specificity of
antibody to HIV in saliva for detection of infection are
between 95% and 100% .www.indiandentalacademy.com

• Several salivary and oral fluid tests have been
developed for HIV diagnosis.
• Orasure® is a testing system that is commercially
• available in the United States and can be used for the
diagnosis of HIV.
• The test relies on the collection of an oral
mucosal transudate (and therefore IgG antibody).
• IgG antibody to the virus is the predominant type of
anti-HIV immunoglobulin

Malignancy
• Salivary analysis may aid in the early detection of certain
malignant tumors.
• p53 is a tumor suppressor protein which is produced in
cells exposed to various types of DNA-damaging stress.
• Inactivation of this suppressor through mutations and
gene deletion is considered a frequent occurrence in the
development
of human cancer
• Abs to this inactive p53 are found in sera & saliva of pts
with tumors
• P53 abs seen in saliva of oscc.www.indiandentalacademy.com

• Defensins are peptides which possess antimicrobial and
cytotoxic properties.
• They are found in the azurophil granules of pmn
• Elevated levels of salivary defensin-1
were found to be indicative of the presence of oral SCC.
• Higher concentrations of salivary defensin-1 were detected
in patients with oral SCC
• A high-positive correlation was observed between
salivary defensin-1 levels and serum levels of SCC-related
antigen www.indiandentalacademy.com

• salivary nitrate, nitrite, and nitrosamine may be
related to the development of oral and gastric cancer
• Increased consumption of dietary nitrate and nitrite
is associated with elevated levels of salivary nitrite.
• Higher levels of salivary nitrate and nitrite, and
increased activity of nitrate reductase, were found in
oral cancer patients compared with healthy
individuals, and were associated with an increased
odds ratio for the risk of oral cancer

• elevated levels of tumor markers c-erbB-2 (erb) and
cancer antigen 15-3 (CA15-3 were found in the saliva of
women diagnosed with breast carcinoma, as compared
with patients with benign lesions and healthy controls
• CA 125 is a tumor marker for epithelial ovarian cancer.
Elevated salivary levels of CA 125 were detected in
patients with epithelial ovarian cancer as compared with
patients with benign pelvic masses and healthy controls.
A positive correlation was found between salivary and
serum levels of CA 125.

Hormone levels
• The majority of hormones enter saliva by passive
diffusion across the acinar cells.
• Salivary hormone levels represent the non-protein-
bound (free) serum hormone levels.
• Small molecules enter by ultra filtration
• Some steroid hormones can be metabolized in the
salivary epithelial cells by intracellular enzymes during
transcellular diffusion, which can affect the availability
of these hormones in salivawww.indiandentalacademy.com

• Due to their lipid solubility, steroid hormones can be
detected in saliva.
• Salivary cortisol levels demonstrate excellent correlation
with free serum cortisol levels
• actual salivary cortisol levels are lower than the serum-free
cortisol levels, possibly due to enzymatic degradation in
the salivary epithelial cells during transcellular diffusion
• Salivary cortisol levels were found to be useful in
identifying patients with Cushing's syndrome & Addison's
disease
• monitoring the hormone response to physical exercise and
the effect of acceleration stresswww.indiandentalacademy.com

• Salivary aldosterone levels demonstrated a high
correlation with serum aldosterone levels and increased
aldosterone levels were found in both the serum and
saliva of patients with primary aldosteronism (Conn's
syndrome)
• The sodium (Na) and potassium (K) conc 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, primary aldosteronism

Testosterone and dehydroepiandrosterone have also been
identified in saliva.
Salivary concentrations were found to
1.5-7.5% of the serum concentrations of these hormones

• Estradiol can be detected in saliva in concentrations that are
only 1-2% of serum concentrations.
• These concentrations are similar to the serum concentrations
of free estradiol, which can diffuse into saliva
• Salivary estriol levels showed a very high correlation
with serum levels of free estriol in pregnant women, and
salivary estriol levels were suggested as a means for the
assessment of feto-placental function
• Decreased levels suggested a marker of fetal growth
retardation.

• Salivary progesterone levels showed good correlation
(r = 0.47-0.58) with serum levels during the
menstrual cycle and reflected the free serum
progesterone levels
• Salivary progesterone levels can be useful for the
prediction of ovulation

• Insulin can be detected in saliva, and salivary insulin
levels have been evaluated as a means of monitoring
serum insulin levels.
• A positive correlation between saliva and serum
insulin levels following a glucose tolerance test was
reported for healthy subjects, niddm patients, and
obese non-diabetic patients
• Salivary insulin levels reached maximal values
approximately 30 minutes after the serum levels

limitations of salivary analysis for hormone evaluation.
• mostly lipid-soluble and hormones with small molecular weight
can be detected in saliva.
• For hormones that demonstrate a constant but low salivary-to-
serum ratio, a sufficiently large sample volume or a more sensitive
analysis method is required.
• In addition, many hormones exhibit marked circadian variations.
Therefore, timing of saliva collection may affect the results.
• The salivary flow rate can also affect the concentrations of certain
hormones. An increase in salivary flow rate will usually result in
reduced concentrations of molecules that reach saliva by diffusion

• Changes in salivary flow rate may lead to changes in
salivary pH. This may affect the entry into saliva of
molecules according to their pka
• Hormones in saliva can be degraded, among other ways, by
enzymes native to saliva, enzymes derived from oral micro-
organisms, and enzymes derived from leukocytes that enter
the oral cavity from gingival sulcus.
• These factors have to be considered when saliva is
evaluated as an alternative for the evaluation of serum
hormone levels.

Drugs
• Passive diffusion across a concentration gradient is thought
to be the major mechanism to account for the appearance
of a drug in saliva.
• Therefore, drugs which are not ionizable, or are not ionized
within the pH range of saliva, are the most suited to
salivary drug monitoring.
• Due to their size, serum binding proteins do not cross the
membrane. Therefore, only the unbound fraction of the
drug (pharma active) in serum is available for diffusion into
saliva

• Saliva is also useful for the monitoring of anti-epileptic
drugs.
• Salivary carbamazepine levels were found to be 38% of
serum carbamazepine levels, and a positive correlation
(r = 0.89) between salivary and serum carbamazepine
levels was observed.
• salivary levels of phenobarbital and phenytoin
demonstrated excellent correlations with serum levels
of these medications

• Cyclosporine is a neutral lipophilic molecule that enters
saliva mostly by passive diffusion, and salivary levels of
this drug reflect the serum levels of free cyclosporine.
• Therefore, salivary cyclosporine levels may correlate
better with serum levels of free, rather than total,
cyclosporine
• Similarly, salivary theophylline concentration
demonstrated a better correlation with serum
concentration of free theophylline (r = 0.85) than with
serum concentration total theophylline

• Therapeutic drugs:
• Antipyrine Metoprolol
• Oxprenolol
• Paracetamol
• Phenytoin
• Primidone
• Procainamide
• Quinine
• Sulfanilamide
• Theophylline
• Tolbutamide
•Caffeine
•Carbamazepine
•Cisplatin
•Cyclosporine
•Diazepam
•Digoxin
•Ethosuximide
•Irinotecan
•Lithium
•Methadone

• Saliva can be used for evaluation of illicit drug use
• Following drug use the appearance of the
drug in saliva follows a time course that is similar to that
of serum. In contrast, drugs appear at a later time point
in urine.
• Presence of drug and not their conc is found
• Exception is ethanol.
• Ethanol is not ionized in serum, is not proteinbound, and,
due to its low molecular weight and lipid solubility,
diffuses rapidly into saliva.

• Consequently, the saliva to- serum ratio is generally
about 1
• Salivary ethanol concentration may be used as
an index of the blood ethanol concentration,
provided that the salivary sample is obtained at least
20 min following ingestion.
• This will allow for absorption and distribution
of alcohol, and prevent a falsely elevated reading due
to the oral route of consumption

• Other recreational drugs that can be identified in saliva
are
• amphetamines, barbiturates,
• benzodiazepines, cocaine,
• Phencyclidine (PCP), and opioids
• Saliva can also be used to detect recent marijuana use
by means of radiommunoassay
• D9-Tetrahydrocannabinol (D9-THC), a major
psychoactive component of marijuana, can be detected
in saliva for at least 4 hours after marijuana is smokedwww.indiandentalacademy.com

• Furthermore, saliva can be used to monitor
tobacco smoking and exposure to tobacco smoke.
• The major nicotine metabolite cotinine was investigated as
an indicator of exposure to tobacco smoking.
• Cotinine is tobacco-specific and has a relatively
long half-life compared with nicotine
• Salivary cotinine levels were found to be indicative of active
and passive smoking
• Salivary thiocyanate was also found to be an indicator of
cigarette smoking

Saliva in forensics
• Salivary test have been used for a wide variety of forensic
studies.
• Samples can be obtained from drinking glasses, cigarette
butts, envelopes, and other sources and then used to
detect blood-group substances or salivary genetic proteins
(primarily proline-rich protein polymorphisms).
• Approximately 85% of individuals secrete blood-group
antigens in their saliva including A, B, H, and Lewis antigens
that have been used for identification of individuals in both
criminal cases and paternity law suits

• Saliva is often present at crime scenes, along with
other body fluids, and since DNA is relatively stable in
the dry state, these samples can be used to place an
individual at the scene of a crime.

Conclusion
• Saliva offers an alternative to serum as a biologic fluid
that can be analyzed for diagnostic purposes.
• Saliva is particularly useful for qualitative (detection of
the presence or absence of a marker) rather than
quantitative diagnosis, which makes it an important
means for the detection of viral infection (especially HIV
due to the non-invasive method of collection), past
exposure and immunity, and the detection of illicit drug
use www.indiandentalacademy.com

• Early studies using saliva as a diagnostic fluid were
hampered by
• lack of understanding on mech by which markers enter oral
cavity
• the difficulty in detection due to low levels
• lack of attention to the method of collection
• storage of samples prior to analysis
These challenges have met as a result of
• careful studies of salivary gland physiology,
• development of sensitive amplification methods (e.g. ELISA,
qRT-PCR), and

• Salivary proteome: The proteome is the protein
complement of the genome
• proteomics is analysis of the portion of the genome
that is expressed.
• The proteomes in body fluids are valuable due to their
high clinical potential as sources of disease markers.
• Collectively, 1166 salivary proteins have been
identified: 914 from the parotid fluid and 917 from the
combined submandibular and sublingual fluids

• Salivary transcriptome: Salivary transcriptome is an
emerging concept.
• They included mRNA molecules that cells use to convey
the instructions carried by DNA for subsequent protein
production.
• This discovery presented a second diagnostic alphabet in
saliva and opened a door to another avenue of salivary
transcriptomic diagnostics
• Other research groups, particularly from forensic
sciences, are focusing on multiplex mRNA profiling for
the identification of body fluids, including saliva

• potential advantages of salivary analysis for the diagnosis
of systemic disease suggest that further studies are
warranted.

References :
• Tina Pfaffe,1 Justin Cooper-white,2,3 Peter Beyerlein.
Diagnostic Potential Of Saliva : Current State And
Future Applications. Clinical Chemistry 57:5 675–687
(2011)
• Eliaz Kaufman And Ira B. Lamster. The Diagnostic
Applications Of Saliva-- A Review. Crit Rev Oral Biol
Med. 13(2):197-212 (2002)

Scope of saliva as a diagnostic fluid/prosthodontic courses

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