Saliva as a Diagnostic Tool

S A L I VA
A S A
D I A G N O S T I C F L U I D
D R A M I T H A G
D E P T O F O R A L A N D M A X I L L O FA C I A L PAT H O L O G Y

CONTENTS :
• Definition
• General properties
• Composition
• Function of saliva
• Formation of saliva
• Method for collecting saliva
• Advantages
• Limitations
• Analysis of saliva done for the diagnosis of systemic disease

Definition:
by Stedmann’s & Lipincott medical dictionary.
A clear, tasteless, odourless, slightly acidic (pH 6.8) viscous fluid,
consisting of the secretion from the parotid, sublingual, submandibular
salivary glands and the mucous glands of the oral cavity.

General properties
• Volume: 1000 to 1500 mL of saliva is secreted per day and, it is
approximately about 1 ml/ minute.
• Contribution by each major salivary gland is:
i. Parotid glands: 25%
ii. Submandibular glands: 70%
iii. Sublingual glands: 5%.
• Reaction: Mixed saliva from all the glands is slightly acidic with pH of
6.35 to 6.85.
• Specific gravity: It ranges between 1.002 and 1.012.
• Tonicity: Saliva is hypotonic.

Salivary flow
• The average person produces approximately 0.5 L – 1.5 L per day
Unstimulated Flow (resting salivary flow―no external stimulus)
• Typically 0.2 mL – 0.3 mL per minute
Stimulated Flow (response to a stimulus, usually taste, chewing, or
medication [eg, at mealtime])
• Typically 1.5 mL – 2 mL per minute

COMPOSITION
saliva contains 99.5% water and 0.5%solids.

FUNCTIONS OF SALIVA
1. PROTECTION
• Fluid nature of saliva provides a washing action that flushes away non adherent bacteria
and other debris.
• Helps in clearance of sugars from the mouth limits their availability to acidogenic plaque
microorganisms.
• Mucins and other glycoproteins provide lubrication, preventing the oral tissues from
adhering to one another and allowing them to slide easily over one another.
• Mucins also form a barrier against noxious stimuli, microbial toxins, and minor trauma.
Ten Cate’s Oral Histology

2. BUFFERING
• Bicarbonate and phosphate ions provide a buffering action that helps to
protect the teeth from demineralization caused by bacterial acids
produced during sugar metabolism.
• Some basic salivary proteins also may contribute to the buffering action
of saliva.
• The metabolism of salivary proteins and peptides by bacteria produces
urea and ammonia, which help to increase the pH.

3. PELLICLE FORMATION
• Many of the salivary proteins bind to the surfaces of the teeth and oral
mucosa, forming a thin film, the salivary pellicle.
• Proteins bind calcium and help to protect the tooth surface.
• Others have binding sites for oral bacteria, providing the initial attachment
for organisms that form plaque.

4. MAINTENANCE OF TOOTH INTEGRITY
• Saliva is supersaturated with calcium and phosphate ions.
• The solubility of these ions is maintained by several calcium- binding
proteins, especially the acidic proline-rich proteins and statherin.
• At the tooth surface the high concentration of calcium and phosphate results
in a posteruptive maturation of the enamel, increasing surface hardness and
resistance to demineralization.
• Remineralization of initial caries lesions can occur; this is enhanced by the
presence of fluoride ions in saliva.

5. ANTIMICROBIAL ACTION
• Saliva contains a spectrum of proteins with antimicrobial activity such as the lysozyme,
lactoferrin, peroxidase, and secretory leukocyte protease inhibitor.
• A number of small peptides include α-defensins and β-defensins, cathelicidin-
LL37,histatins that function by inserting into membranes and disrupting cellular or
mitochondrial functions are present in saliva.
• These proteins and peptides exhibit antibacterial, antifungal and antiviral activity.
• Secretory immunoglobulin A (IgA), causes agglutination of specific microorganisms,
preventing their adherence to oral tissues and forming clumps that are swallowed.

6. TISSUE REPAIR
• A variety of growth factors and other biologically active peptides and
proteins are present in small quantities in saliva.
• Many of these substances promote tissue growth and differentiation,
wound healing.

7. DIGESTION
• The solubilization of food substances and the actions of enzymes such as
amylase and lipase begin the digestive process.
• The moistening and lubricative properties of saliva also allow the formation
and swallowing of a food bolus.

8. TASTE
• Saliva solubilize food substances so that they can be sensed by taste
receptors located in taste buds.
• Saliva produced by minor glands in the vicinity of the circumvallate
papillae contains proteins that are believed to bind taste substances and
present them to the taste receptors.

Formation of saliva occurs in 2 stages.
Stage 1 : Production of primary saliva from the cells of
secretory end pieces & intercalated ducts, which is an
isotonic fluid
Stage 2 : The primary saliva is modified as it passes through
the striated & excretory ducts mainly by reabsorption &
secretion of electrolytes.
The final saliva that reaches the oral cavity is hypotonic.
FORMATION AND SECRETION OF SALIVA

Formation of saliva
• acinar cells produce initial saliva composed of water, ions, enzymes, and
mucus
• initial saliva is isotonic and has approximately same osmolarity as plasma
• initial saliva passes from acinus to duct
• ductal cells modify initial saliva to produce final saliva by altering various
electrolyte concentrations
• ductal cells absorb Na+ and Cl-, decreasing ion concentrations in saliva
• ductal cells secrete K+ and HCO3
-, increasing ion concentrations in saliva
• salivary HCO3
- neutralizes oral bacterial acids
• maintains dental health
• final saliva is hypotonic and has lower osmolarity than plasma
http://www.medbullets.com/step1-gastrointestinal/10023/salivary-secretion

Regulation of salivary secretion
salivary secretion is stimulated by both sympathetic and parasympathetic nervous systems
• sympathetic nervous system
• sympathetic input to salivary glands originates in thoracic segments T1-T3 with preganglionic nerves
that synapse in superior cervical ganglion
• postganglionic sympathetic neurons release norepinephrine (NE)
• ↑ NE → stimulates β-adrenergic receptors → stimulates adenylyl cyclase → ↑ cAMP → ↑ saliva
secretion
• parasympathetic nervous system
• parasympathetic input to salivary glands originates in facial nerve (CN VII) and glossopharyngeal nerve
(CN IX)
• postganglionic parasympathetic neurons release ACh
• ↑ ACh → stimulates muscarinic receptors → ↑ IP3 and Ca2+ → ↑ saliva secretion
parasympathetic activity stimulated by food, smell, nausea, and conditioning
parasympathetic activity inhibited by dehydration, fear, and sleep
http://www.medbullets.com/step1-gastrointestinal/10023/salivary-secretion

FORMATION AND SECRETION OF SALIVA
• Protein secretion occurs by exocytosis, that is, the fusion of
secretory granules with the luminal membrane to release
their contents into the lumen.
• The binding of the sympathetic transmitter norepinephrine
(NE) to β-adrenergic (β) receptors on the basolateral
membrane activates a heterotrimeric G-protein (Gs), which
in turn activates adenylyl cyclase (AC), catalyzing the
formation of cyclic adenosine monophosphate (cAMP)
from adenosine triphosphate (ATP).
• Cyclic adenosine monophosphate activates protein kinase A
(PKA), which phosphorylates other proteins in a cascade
leading to exocytosis.
Ten Cate’s Oral Histology

• Fluid and electrolyte secretion is stimulated mainly by the
binding of the parasympathetic transmitter, acetylcholine
(ACh) to muscarinic cholinergic (MC) receptors and also
by norepinephrine binding to α-adrenergic receptors (α).
• These receptors activate a heterotrimeric G-protein
(Gq/11), causing activation of phospholipase C (PLC),
which converts phosphatidylinositol bisphosphate (PIP2)
to inositol trisphosphate (IP3) and diacylglycerol (DAG).
• Inositol trisphosphate causes the release of Ca2+ from
intracellular stores, probably the endoplasmic reticulum.
The increased Ca2+ concentration opens Cl– channels in
the luminal membrane and K+ channels in the basolateral
membrane and activates the basolateral Na+/K+/2Cl–
cotransporter.

• The increased luminal Cl– is balanced by the movement of
extracellular Na++ across the tight junctions (TJ), and the
resulting osmotic gradient pulls water into the lumen
through the cell via the water channel aquaporin 5 and
through the tight junction.
• The basolateral Na+/K+/2Cl– cotransporter and the
Na+/K+–adenosine triphosphatase serve to maintain the
intracellular electrolyte and osmotic balances.
• Calcium also stimulates exocytosis, but to a lesser extent
than cyclic adenosine monophosphate, and it modulates the
activity of protein kinase A and protein kinase C (PKC).
Protein kinase C, in turn, modulates exocytosis and
intracellular Ca2+ concentrations.

Instructions for Collecting Saliva:
1.Remove cap from cryovial
2.Remove Saliva Collection Aid from packaging and place securely into cryovial.
3.Instruct participants to allow saliva to pool in the mouth.
4.With head tilted forward, participants should drool through the SCA to collect saliva in
the cryovial.
5.Repeat until sufficient sample is collected. Reserve air space in the vial to accommodate
the expansion of saliva during freezing. Collection of samples to be analyzed for multiple
analytes may require additional cryovials.
6.Replace cap onto cryovial.
How to Collect Saliva: Collection Methods and Devices
Saliva Collection and Handling Advice 2nd Edition Copyright 2011, Salimetrics, LLC

The Salimetrics Oral Swab (SOS)
• If participants are not willing or able to drool saliva into a vial.
• If the saliva samples are to be analyzed for cortisol, testosterone, α-amylase,
chromogranin A, C-reactive protein, or SIgA, the Salimetrics Oral Swab (SOS) is an
excellent alternative to passive drool because of its ease of use.
• The SOS also helps filter mucus and other matter from the sample, which may help
improve immunoassay results.

Collection Methods and Devices:
Infants and Small Children
The Children’s Swab and Infant’s Swab
We recommend the SalivaBio Children’s Swab (SCS) for children under the age of 6,
and the SalivaBio Infant’s Swab (SIS) for infants under 6 months of age.
The extra-length SalivaBio Children’s Swab (SCS) may also be used for saliva collection
from infirmed adult patients to avoid any danger of choking.

All SalivaBio swabs are made from the same non-toxic, inert polymer which is
guaranteed for consistency across all lots, making it ideal for longitudinal and multi-
participant group studies.
These devices are manufactured in longer lengths and narrower widths to allow one end
of the swab to be held by a parent or technician while the other end is placed in the
child’s mouth.
The diameters are appropriate for the size of children’s mouths.
The polymer material is durable and can withstand chewing, and its taste and texture are
also acceptable to children.
The volume of sample recovered from the SCS and SIS is typically in the range of 200-
1000 μL. Like the adult SOS, samples collected with either the SCS or the SIS may be
tested for various analytes

• Passive drool is highly recommended because it is both cost effective and approved for
use with almost all analytes.
• If participants are not willing or able to drool saliva into a vial, the SalivaBio Oral Swab
may be used as an alternative collection method, but only for certain analytes
• To avoid problems with analyte retention or the introduction of contaminants, use only
high quality polypropylene vials for collection, such as 2 ml cryovials.
• The vials used must seal tightly, must be able to withstand temperatures down to -80oC,
and must be externally threaded to allow for use of the Saliva Collection Aid to
effectively guide drool directly into the cryovial.
Saliva Collection and Handling Advice 2nd Edition Copyright 2011, Salimetrics, LLC

Advantages
1. Noninvasive diagnosis of disease and monitoring of general health.
2. Painless, patient suffers no discomfort and little anxiety in the collection process.
3. Simple in collection with a modest trained assistant and applicable in remote
areas.
4. Relatively cheap technology as compared to other tests.
5. Cost effective applicability for screening large population.

6. Can be used to study special population where blood sampling is a problem e.g
children, anxious /handicap/ elderly patients.
7. Convenient for multisampling.
8. Safer for health professionals than blood tests.
9. Compared to blood and urine, saliva is also cheaper to store and ship.
10. In addition saliva does not clot and can be manipulated more easily than blood.
IOSR Journal of Dental and Medical Sciences (IOSR-JDMS) e-ISSN: 2279-0853, p-ISSN: 2279-0861. Volume 11, Issue 6 (Nov.- Dec.
2013), PP 96-99 www.iosrjournals.org Saliva- A Diagnostic Tool. Dr Vijaylaxmi B Madalli, Dr Shrinivas M Basavaraddi,Dr Krishna
Burde, Dr Priya Horatti

Limitations:
1. Levels of certain markers in saliva are not always a reliable reflection of the levels of
these markers in serum.
2. Salivary composition can be influenced by the method of collection and degree of
stimulation of salivary flow.
3. Changes in salivary flow rate may affect the concentration of salivary markers and
also their availability due to changes in salivary pH.
4. Variability in salivary flow rate is expected between individuals and in the same
individual under different conditions.

6. In addition, many serum markers can reach whole saliva in an unpredictable way
(i.e. gingival cervical fluid flow and through oral wounds). These parameters will
affect the diagnostic usefulness of many salivary constituents.
7. Furthermore, certain systemic disorders, numerous medications and radiation may
affect salivary gland function and consequently the quantity and composition of
saliva.
8. Whole saliva also contains proteolytic enzymes derived from the host and from oral
microorganisms. These enzymes can affect the stability of certain diagnostic
markers. Some molecules are also degraded during intracellular diffusion into
saliva.
IOSR Journal of Dental and Medical Sciences (IOSR-JDMS) e-ISSN: 2279-0853, p-ISSN: 2279-0861. Volume 11, Issue 6 (Nov.- Dec.
2013), PP 96-99 www.iosrjournals.org Saliva- A Diagnostic Tool. Dr Vijaylaxmi B Madalli, Dr Shrinivas M Basavaraddi,Dr Krishna
Burde, Dr Priya Horatti

Analysis of saliva done for the diagnosis of following
Hindawi Publishing Corporation. ISRN Dentistry. Volume 2014, Article ID 158786, 8 pages. http://dx.doi.org/10.1155/2014/158786 .
Review Article . Salivary Diagnostics: A Brief Review. Narasimhan Malathi,1 Sabesan Mythili,1 and Hannah R. Vasanthi2

Potential Biomarkers in Saliva
1. Autoimmune Diseases
A. Sjogren’s Syndrome (SS)
• It is an autoimmune disorder characterized by reduced secretion of the salivary glands and
lacrimal glands and associated endocrine disturbance. Sialochemistry offers great value in
the diagnosis of SS.
• An increase in the levels of immunoglobulins, inflammatory mediators, albumin, sodium,
and chloride and a decrease in the level of phosphate are indicative of SS.
• Salivary protein analysis demonstrated an increased level of lactoferrin, beta 2
microglobulin, lysozyme C, and cystatin C.
• The levels of salivary amylase and carbonic anhydrase were decreased.

B. Multiple Sclerosis
• Multiple sclerosis (MS) is an inflammatory disease characterized by loss of
myelin and scarring caused due to destruction/failure of myelin producing
cells by the immune system.
• Salivary diagnostics shows no significant change in the saliva of patients
with multiple sclerosis except for a reduction in IgA production

C. Sarcoidosis
Sarcoidosis is an inflammatory disease of the lymph nodes, lungs, liver, eyes,
skin, or other tissues.
Salivary diagnostics demonstrates a decrease in the secretion volume of saliva
in addition to a reduction in the enzyme activity of alpha-amylase and
kallikrein in most of these patients.
However, there was no correlation between the decrease in the enzyme activity
and the secretion volume

2. Dental Caries and Periodontal Diseases.
• The increased numbers of Streptococcus mutans and lactobacilli in saliva have been
associated with increased caries prevalence and root caries.
• Periodontal diseases have been associated with increased levels of aspartate
aminotransferase (AST) and alkaline phosphatase (ALP).
• Salivary AST can be used as a marker for monitoring periodontal disease.
• Lower levels of uric acid and albumin were associated with periodontitis and diabetes.
This could be attributed to the oxidative stress present in the oral cavity during these
conditions.
• In patients with type 2 diabetes mellitus, the salivary expression of pIgR, Arp 3, CA VI,
and IL-1Ra was down- regulated, whereas PLS-2, LEI, and IGJ chain appeared to be
upregulated
Diagnostic Applications of Saliva in Dentistry 1Prabhakar AR, 2Akanksha Gulati, 3Deepak Mehta, 4Sugandhan S

3. Malignancy
• Tang et al. - lncRNA , p53 protein and miRNAs, - oral squamous cell carcinoma
(OSCC)
• Salivary lactate dehydrogenase, Salivary cortisol, salivary nitrate and nitrite levels
increased - oral cancer
• Salivary adenosine deaminase (ADA) activity is significantly increased in squamous
cell carcinoma of the tongue progressively from stage I to stage III.
• Fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1)
concentrations in saliva are significantly elevated - salivary gland tumors
• Salivary mRNA biomarkers (CCNI, EGFR, FGF19, FRS2, CA 125 and GREB1) - lung
cancer
• AGPAT1, B2M, BASP2, IER3, and IL1B, CA 125 - ovarian cancer
• CA15-3, CA 125 - breast cancer.

4. Bone Turnover Markers:
• Saliva can be used in mass screening for metabolic bone disorder.
• Human saliva was analysed and there is a significant correlations have been reported
between age, body mass index, deoxypyridinium (D-PYR) and osteocalcin (OC)
concentration and calcaneus T scores.
• This suggests that saliva could be used as a fluid for assay of human biomarkers of bone
turnover.
• Scannapieco et al. noted a positive association between alveolar bone loss and salivary
concentrations of hepatocyte growth factor and interleukin-1 beta. However, there was a
negative association between alveolar bone loss and salivary osteonectin.
• The increased levels of alkaline phosphatase (ALP) activity in periodontitis have been
correlated with the alveolar bone loss

5. Drug Level Monitoring
• Saliva is used to detect the presence of nicotine to monitor exposure to tobacco smoke,
cannabinoids, cocaine, phencyclidine, opioids, barbiturates, diazepines, amphetamines, and
ethanol.
• In drug level monitoring, only the unbound fraction of the drug in serum diffuses into the
saliva and is detectable in the saliva.
• Rapid detection of illicit drug use can also be done through the direct analysis of
methamphetamine, cocaine, and 3,4-methylenedioxymethamphetamine in saliva by a
hydrophobic porous silicon array.
• Saliva is a biological matrix used for drug testing of 𝛾- hydroxybutyric acid (GHB) levels
because of its merit of easy, noninvasive collection and stability of the drug. Also, the values
of the drug in saliva correlate with its level in the blood

6. Genetic Disorders
A. Cystic Fibrosis:
• Cystic fibrosis (CF) is a genetically determined condition which is caused due to a
mutation in the CFTR gene.
• The CFTR protein is expressed in the epithelial cells of the parotid gland causing
parotid gland involvement.
• The level of activity of cathepsin-D in saliva is significantly higher
• The values of sodium, potassium, and chloride concentrations is significantly higher
• Salivary calcium concentration, magnesium concentration, and lactate dehydrogenase
levels is increased.

B. Ectodermal Dysplasia:
• The most common form of ectodermal dysplasia is the X-linked hypohidrotic
ectodermal dysplasia (HED).
• Lexner et al. performed a study on whole saliva flow and composition in males
affected by HED and the female carriers.
• He found that there was reduced whole saliva flow and the concentration of
inorganic constituents and total protein was high.
• However, the activity and the concentration of the alpha-amylase in the saliva were
reduced

7. Cardiovascular Diseases.
• Salivary markers of cardiovascular diseases include C-reactive protein (CRP), myoglobin (MYO),
creatinine kinase myocardial band (CK- MB), cardiac troponins (cTn), and myeloperoxidase, which, when
used in combination with an ECG, shows a positive correlation with myocardial infarct patients as
compared to healthy controls.
• The salivary MYO levels are significantly higher within 48 h of onset of chest pain in acute myocardial
infarction (AMI) patients.
• In a study performed by Miller et al.,
In AMI they found that the salivary concentrations of CRP, TNF-𝛼, and MMP-9 were higher
salivary myeloperoxidase and salivary soluble ICAM-1levels elevated
salivary soluble CD40 ligand is lower
• Increased levels of salivary lysozyme have been shown to be associated with hypertension, an early stage
of cardiovascular disorders

8. Infections
(1) Viral infections : Measles virus-specific IgM,
HIV—HIV-1, HIV-2—antibodies, salivary proteins
(2) Bacterial infections : Mycobacterium tuberculosis, MUC 5B, and MUC 7
(3) Fungal infections : Candidiasis immunoglobulins, Hsp 70, and calprotectin,
histatins, mucins, basic proline rich proteins, and peroxidases

9. Psychological research
Salivary amylase, cortisol, substance P, lysozyme, secretory IgG, and
testosterone
10. Renal diseases
Cortisol, nitrite, uric acid, sodium chloride, pH, alpha-amylase, and
lactoferrin. Salivary phosphate, serum creatinine, and glomerular
ltration rate
11. Diseases of the adrenal cortex
Salivary cortisol
Dent Clin North Am. 2011 January ; 55(1): 159–178. doi:10.1016/j.cden.2010.08.004. Saliva as a Diagnostic Fluid . Daniel
Malamud, PhDa,b and Isaac R. Rodriguez-Chavez, PhDc

Conclusion
Blood is still the gold standard for diagnostics of diseases and drugs, Saliva offers an alternative to serum
as a biologic fluid for diagnostic purposes
The greatest mile- stone in salivary diagnostics is to identify the disease biomarkers and to transfer it
from the laboratory to the clinical practice.
But the growth of salivary diagnostics has been hindered because of lack of sensitive detection methods,
lack of correlation between the biomolecules in the blood and saliva, and the circadian variations in
saliva.
However, unlike blood and other body fluids, salivary diagnostics offers an easy, inexpensive, painless,
and stress free approach to disease detection.

Reference :
• Shafers textbook of oral pathology
• Tencate’s Oral histology
• Hindawi Publishing Corporation. ISRN Dentistry. Volume 2014, Article ID 158786, 8 pages.
http://dx.doi.org/10.1155/2014/158786 . Review Article . Salivary Diagnostics: A Brief Review. Narasimhan
Malathi,1 Sabesan Mythili,1 and Hannah R. Vasanthi2
• Saliva Collection and Handling Advice 2nd Edition Copyright 2011, Salimetrics, LLC
• Diagnostic Applications of Saliva in Dentistry . 1Prabhakar AR, 2Akanksha Gulati, 3Deepak Mehta, 4Sugandhan S
• Saliva as a diagnostic tool for oral and systemic diseases . Mohammad A. Javaid a, Ahad S. Ahmed b, Robert
Durand c, Simon D. Tran d,*
• Dent Clin North Am. 2011 January ; 55(1): 159–178. doi:10.1016/j.cden.2010.08.004. Saliva as a Diagnostic Fluid.
Daniel Malamud, PhDa,b and Isaac R. Rodriguez-Chavez, PhDc
• IOSR Journal of Dental and Medical Sciences (IOSR-JDMS) e-ISSN: 2279-0853, p-ISSN: 2279-0861. Volume 11,
Issue 6 (Nov.- Dec. 2013), PP 96-99 www.iosrjournals.org Saliva- A Diagnostic Tool. Dr Vijaylaxmi B Madalli, Dr
Shrinivas M Basavaraddi,Dr Krishna Burde, Dr Priya Horatti

Saliva as a Diagnostic Tool

More Related Content

What's hot

Similar to Saliva as a Diagnostic Tool

More from oral and maxillofacial pathology

Recently uploaded

Saliva as a Diagnostic Tool

Editor's Notes