SALIVA

By,

Harshavardhan Patwal

TOPICS AND HEADINGS COVERED

• SALIVA

– COMPOSITION AND ORIGIN

• NORMAL PHYSIOLOGIC FUNCTIONS IN PERIODONTAL

HOMEOSTASIS

• COLLECTION OF SALIVA

• USE OF SALIVA AS A DIAGNOSTIC FLUID FOR PERIODONTAL

DIAGNOSIS

SALIVA

• Saliva is a clear, slightly acidic mucoserous exocrine secretion

• Whole saliva is a mixture of fluids from major and minor

salivary glands , gingival crevicular fluid , that also contains

bacteria and food debris

SALIVARY GLANDS

• These are exocrine glands that are responsible for the

secretion of saliva

• The salivary glands are categorized as major and minor

• The major salivary glands are the parotid, submandibular and

sublingual glands

• Minor glands that produce saliva are located in the lower lip,

tongue, palate, cheeks and pharynx

• The major salivary glands contribute to maximum saliva production.

The average daily flow of saliva is between 1 to 1.5 liters in healthy

individuals. Percentage contribution from different salivary glands are

as follows:

• Parotid gland: 20%

• Submandibular:65%

• Sublingual: 7 to 8%

• Minor salivary glands: 10%

These percentages are described for unstimulated whole saliva.

Stimulated high flow rates change the percentage contribution from

different salivary glands with the maximum from parotid gland:50%

ANATOMICAL LOCATION OF THE SALIVARY GLANDS

CELLS OF THE SALIVARY GLAND SYSTEM

The various cells in the salivary glands are:

Acinar cells: which are responsible for production of saliva.

These cells are responsible for the type of salivary secretion

Duct system cells: these are cells that line the ducts which are

categorised as intercalated, striated and excretory

Myoepithelial cells: these are long cells that are wrapped

around the acinar cells and contract on stimulation to squeeze

out the salivary secretions

MORPHOLOGICAL AND HISTOLOGICAL ASPECTS OF MAJOR SALIVARY GLANDS

PAROTID SUBMANDIBULAR SUBLINGUALGLAND TYPE COMPOUND ACINAR COMPOUND

TUBULOACINARCOMPOUND TUBULOACINAR

POSITION MANDIBULAR RAMUS

BENEATH MANDIBLE FLOOR OF THE MOUTH

SIZE LARGEST INTERMEDIATE SMALLESTMAIN DUCT STENSONS WHARTONS BARTHOLINSINTERLOBULAR DUCTS

LINED WITH PSEUDOSTRATIFIED COLUMNAR EPITHELIUM

SAME SAME

STRIATED DUCTS SINGLE LAYER CUBOIDAL TO COLUMNAR CELLS

SAME DUCTS ARE RARE

INTERCALATED FLAT TO CUBOIDAL SAME BUT SHORT DUCTS ARE RARETERMINAL SECRETORY UNITS

SEROUS ACINI SEROUS ACINI , MUCOUS ACINI WITH SEROUS DEMILUNES

MUCOUS ACINI WITH DEMILUNES

ROLE OF THE DUCT SYSTEM CELLS

• Intercalated duct cells: they are the first duct network

connecting acinar secretions to the rest of the gland

• Striated duct cells: they function for electrolyte regulation in

resorbing sodium

• Excretory duct cells: help in continuing sodium resorption and

potassium secretion

COMPOSITION OF HUMAN SALIVA• Saliva is composed of numerous components. it is a very dilute

fluid composed of about 99% water

• Saliva is not considered an ultrafiltrate of plasma. It is initially

isotonic, as it is formed in the acinar cells, but it becomes

hypotonic, when it travels through the duct system.

• The hypotonicity of saliva in an unstimulated state allows the

taste buds to perceive different tastes. hypotonicity during low

flow periods helps in expansion and hydration of mucin

glycoproteins which protect the tissues of the mouth

COMPOSITION

• As previously mentioned, the main content of saliva is water.

The other components are

• Electrolytes: ammonia , bicarbonate, calcium, fluoride,

hypothiocyanate, iodine, magnesium, nitrate, nitrite,

phosphates, potassium, sodium, sulphates and thiocyanates

Organic compounds: – amino acids, – creatinine, – fatty acids,– glucose,– lactic acid, – lipids, – sialic acid, – urea, – uric acid.

Proteins and macromolecules:– aggregins,– albumins, – blood group substances, – cystatins, – enzymes,– fibronectin,– fucose rich gycoprotein, – growth factors, – gustin, – histatins, – immunoglobulins, – kallikerin,– lactoferrin,– mucin rich glycoproteins,– proline rich proteins, – tyrosine rich proteins,– ribonucleases

Whole saliva

Extrinsic Food

Tooth paste and mouth

rinse derivative

Bronchial and nasal secretions

Microbes, viruses, fungi

and mycoplasma

Lining cellsEpithelial keratins

Blood and its derivatives

Inflammatory cells

Salivary gland derivatives

WaterProteins

Electrolytes

SALIVA FLOW• There is a great variability in salivary flow rates in different persons

• The accepted flow rate of unstimulated saliva is anything above.1 ml

per minute. For stimulated saliva, the accepted norm is .2 ml per

minute. Salivary flow is highly individualized and should be recorded.

• On an average, the flow rate for unstimulated saliva is .3 ml per

minute. With the average total of 16 hours of unstimulated flow

being 300 ml.

• Stimulated flow of saliva is 7 ml per minute on an average.

Stimulated saliva is supposed to contribute as much as 80 to 90 % of

the daily secretion.

FACTORS THAT CONTROL SALIVARY FLOW

• Basically controlled by the salivary centre composed of nuclei in the

medulla . The other factors that affect salivary flow rate are

• Act of chewing

• Food constituents such as sweet, salt and bitters

• Olfaction and noxious smell

• Pain

• Stress

• Pharmacologic agents

• Local and systemic diseases that affect the salivary glands

FACTORS AFFECTING SALIVA FOW

• In the same individual, salivary flow has a circadian and circannual

rhythm

• Salivary flow is minimum to zero in the night and peaks during the

day

• In the year low salivary flow rates occur in summer and peak in

winter

• In the mouth, there are areas with high and low flow. The

mandibular lingual areas have high flow while maxillary anteriors

have a low flow rate

BASIC FUNCTIONS OF SALIVA

• Antimicrobial activity

• Buffering capacity

• Clearance of microbes by flow mechanism

• Food preparation and bolus formation

• Food digestion due to the presence of salivary amylase and

lipase

• Formation of intraoral pellicles

• Lubrication and protection of soft and hard tissues of the

mouth

• Post translational molecular processing

• Remineralisation

• Speech

• Taste

• Water and electrolyte balance

ROLE OF SALIVA IN ORAL HEALTHFUNCTION SALIVARY COMPONENTS PROBABLE MECHANISM

LUBRICATION GLYCOPROTEINS, MUCOIDS COATING SIMILAR TO GASTRIC MUCIN

PHYSICAL PROTECTION GLYCOPROTEIN, MUCOIDS COATING SIMILAR TO GASTRIC MUCIN

CLEANSING PHYSICAL FLOW CLEARANCE OF BACTERIA AND DEBRIS

BUFFERING ACTION BICARBONATES AND PHOSPHATES

ANTACID MECHANISM

TOOTH INTEGRITY MAINTANANCE

MINERALS , PELLICLE REMINERALISATION, MATURATION

ANTIBACTERIAL ACTION IG A, LYSOZYME, LACTOPEROXIDASE

CONTROL OF BACTERIAL COLONIZATION, BREAKS CELL WALLS, OXIDATION OF SUSCEPTIBLE BACTERIA

SALIVA AS A DIAGNOSTIC TOOL

• Besides its basic homeostatic functions in the mouth saliva is

a very important diagnostic fluid. The reasons behind the

usefulness of saliva in this regard are:

• Easy to collect

• Most non invasive fluid to collect

• Abundant in quantity to sample

• Does not need costly equipment for sampling

DISADVANTAGES OF SALIVA AS A DIAGNOSTIC FLUID

• IS CONTAMINATED BY GCF, BRONCHIAL AND PHARYNGEAL

SECRETION

• CONTAMINATED BY FOOD DEBRIS AND MICROBES

• AFFECTED BY PHARMACOLOGICAL AGENTS

• DUE TO CIRCADIAN AND CIRCANNUAL VARIATIONS IN

SALIVARY FLOW, THE ASSAYS CANNOT BE PREDICTABLE

SALIVA IN PERIODONTAL DIAGNOSIS

• As discussed earlier, saliva is a protector of the oral cavity and

periodontal structures

• Due to its various constituents saliva is beneficial against

protecting from periodontal disease. However saliva is a

double edged sword due to its promoting effects on biofilm

formation

CHRONIC PERIODONTITIS

• As all of us know, chronic

periodontitis, is an

inflammatory disease of

microbial origin initiated

and perpetuated by

pathogenic bacteria in

dental plaque.

PERIODONTAL DIAGNOSIS

• CLINICAL EXAMINATION

• PERIODONTAL PROBING

• RADIOGRAPHIC EXAMINATION

• ASSAYS OF SERUM, GCF AND SALIVA

• MICROBIOLOGIC ANALYSIS OF PLAQUE BIOFILM

IMPORTANCE OF ASSAYS OF SALIVA

• Saliva based assays of various biomarkers are helpful because

• They help in understanding the full mouth status

• They reflect the role of any systemic changes

• They help in assessing disease activity and periodontal

disease progression

HOW TO COLLECT SALIVA

• Before understanding the various biomarkers in saliva and the

assay methods, we should understand how to collect and

sample saliva. But there are certain limitations we should

understand during saliva collection

• Saliva wets and lubricates the oral surfaces. Collection of

whole saliva means gathering the fluid that will flow outside

the mouth, which is not the entire amount of saliva since a

part of it will still remain in the mouth.

• Collection of minimal or less amounts of saliva is a feature of

salivary hypofunction that will hamper assay techniques

• Salivary fractions are lost in the collection process owing to

evaporation and retention of the fluid in collection devices

• Saliva secretion varies during the day and is influenced by

temperature, season, hydration status, mood and systemic

condition. thus the same individual will probably yield diverse

secretory rates at different occasions

• The collection conditions of saliva is not absolute. An

unstimulated state may not be completely devoid of stimuli ,

since minor oral movements may provide stimuli. On the

other hand, its completely not possible to standardize the

intensity of stimulation

• Nevertheless , saliva is an important diagnostic specimen and

its collection and storage should be learned

GENERAL CONSIDERATIONS FOR SALIVA COLLECTION

• Collect saliva generally during the same time of the day. For experimental assays, the preferred time is between 9 and 11 am

• Refrain from eating and drinking at least 90 minutes before sampling

• If applicable stop the drugs that would affect salivary secretion a day prior to sampling

• Rinse mouth with deionised water prior to collection of saliva• Saliva collection period should be a minimum of ten minutes

VARIOUS TYPES OF SALIVA THAT CAN BE SAMPLED

• WHOLE SALIVA

• PAROTID SALIVA

• SUBMANDIBULAR SALIVA

• SUBLINGUAL SALIVA

• SALIVA FROM MINOR SALIVARY GLANDS

WHOLE SALIVA• Whole saliva is a mixture not only of glandular secretions, but

also contains bronchial , nasal secretions, GCF, food debris and cells.

• The various methods of collecting whole saliva have been mentioned– Draining method– Spitting method– Suction method– Absorbent method

DRAINING METHOD

• Saliva is allowed to drain off or drip off the lip into a pre

weighed graduated test tube.

• The subject is instructed to expectorate into the test tube at the

end of the collection period.

• The tube can be fitted with a funnel to ease the collection

process

• The amount of saliva sampled is weighed or measured in the

graduated scale

SPITTING METHOD

• Saliva is allowed to accumulate in the floor of the mouth.

• The patient spits out into a pre weighed tube once in 60

seconds when he develops the urge to expectorate or

swallow.

• When we collect stimulated whole saliva by the spitting

method, the patient is instructed to chew on paraffin wax and

spit the saliva into the tube

SUCTION METHOD

• Saliva is continuously aspirated from the floor of the mouth

into a cup using an aspirator or saliva ejector

• The collected saliva is then weighed

ABSORBENT METHOD• Saliva is collected or absorbed by pre weighed swab, cotton

rolls or gauze squares placed in the mouth at the orifices of

the salivary glands.

• Following this the preweighed swabs or cotton rolls are

reweighed.

• A commercially available absorbent method for the collection

of whole saliva is the salivette method. Using this method, the

saliva is collected by chewing cotton wool which is citric acid

treated. Recovery of the saliva is done by returning the

soaked cotton to the salivette and centrifuging the container

PAROTID SALIVA

• This is the easiest glandular saliva that can be collected. The

orifice of the parotid gland is very easy to cannulate.

• Cannulation technique: in this method, the orifice of the main

stensons duct is checked using a blunt lacrimal probe. Next a

thinner tube can be inserted in the duct via an orifice. If

needed , the tube can be fixed with a suture so that no

leakage will occur

LASHLEY CUP / CARLSON CRITTTENDEN CUP

• These cups are easily applied, even by an untrained

personnel. The inner chamber is attached to a rubber bulb or

a suction device via a plastic tube and the cup is placed over

the stensons duct.

• Parotid saliva is always collected in a stimulated state

SUBMANDIBULAR / SUBLINGUAL SALIVA

• Submandibular / sublingual glands contribute 30 to 60% of

the whole stimulated saliva. The various methods to collect

saliva from these glands are

• Cannulation: secretions from these glands enter the oral

cavity via a common duct, making it difficult to collect

secretions. Tapered polythene tubing may be used for

cannulation of the whartons duct

SEGREGATOR ( INDIVIDUAL PROSTHESIS)

• Schneyer et al have suggested the method of collecting

submandibular and sublingual saliva. The custom made

segregator has a central chamber for collection of

submandibular saliva and 2 lateral chambers for collection of

sublingual saliva

• The collector is placed over the lower jaw and has to be

custom made for the patient

WOLFF APPARATUS• The collection system for submandibular and sublingual saliva

is composed of

• Collection tubing• A buffering chamber• A storing tube• A suction device

COLLECTION OF SALIVA FROM MINOR SALIVARY GLANDS

• Labial and buccal gland saliva

• Saliva from the minor labial and buccal glands is collected by

paper strip method

• Either 2.2 * 4.4 mm periopaper strips or 6*16 mm pure

chromatography paper strips are used and held with cotton

pliers on the sampling site to wet and collect the saliva

PALATINE SALIVA

• Palatine saliva is sampled and collected using

• Micropippetes

• Filter paper

• High purity chromatography paper

• Sialopaper

• Individual collection prosthesis

PROCESSING AND STORAGE OF SALIVA: GENERAL GUIDELINES

• After collection, the sampled saliva should be processed properly and stored. The general guidelines for processing and storage are as follows

• Standardised collection on ice cooled vials• Vortexing for 2 minutes• Centrifugation at 10,000 *g for 5 minutes or 3,000* g for 20

minutes• Storage at – 20 to -80 degrees• The freeze thaw cycle should be done only once during

assaying

SALIVARY BIOMARKERS IN PERIODONTAL DISEASE

• As mentioned previously saliva is an abundantly available fluid

that can be easily collected and sampled for various assays of

biomarkers. The definition of a biomarker is:

• A biochemical feature or facet that can be used to measure

the progress of disease or the effects of treatment.

• A biomarker can also be defined as:

A biological molecule found in blood, other body fluids, or

tissues that is a sign of a normal or abnormal process, or of a

condition or disease. A biomarker may be used to see how

well the body responds to a treatment for a disease or

condition. Also called molecular marker and signature

molecule.

DIAGNOSTIC TESTS TO ASSAY BIOMARKERS

• Biomarkers to asses disease activity and disease progression.

These biomarkers are assayed using various diagnostic tests

• A diagnostic test is said to be superior only if there is both

sensitivity and specificity

SENSITIVITY AND SPECIFICITY TEST RESULT DISEASE NO DISEASE

POSITIVE A ( TRUE POSITIVE) B ( FALSE POSITIVE)

NEGATIVE C ( FALSE NEGATIVE) D ( TRUE NEGATIVE)

SENSITIVITY A/ A+C

SPECIFICITY D/B+D

POSITIVE PREDICTIVE VALUE

A/A+B

NEGATIVE PREDICTIVE VALUE

D/C+D

• The sensitivity of any test is the proportion of subjects with

disease who test positive for the disease

• The specificity of any test is the proportion of subjects

without the disease who test negative for the disease

POSITIVE AND NEGATIVE PREDICTIVE VALUE

• The possibility that a person tested positive for a test has the

disease is called positive predictive value

• The possibility that a person tested negative for the disease

not having the disease is called negative predictive value

THE SALIVARY BIOMARKERS IN CHRONIC PERIODONTITIS

• The various salivary biomarkers useful for periodontitis diagnosis can be sub classified as follows

• Enzymes• Immunoglobulins• Proteins• Phenotypic markers• Host cells• Ions• Hormones• Bacteria• Volatile sulfur compounds

THE ENZYME BIOMARKERS IN SALIVA

• Alpha glucosidase• Alkaline phosphatase• Aminopeptidases• Beta galactosidase• Beta glucosidase• Beta glucoronidase• Caprylate esterase

lipase• Collagenase

• Elastase• Esterase• Gelatinase• Kallikerin• Kininase• Lysozyme• Myeloperoxidase• trypsin

ENZYMES IN SALIVASOURCES

The various sources of enzymes that are found in saliva are

( Chauncey et al, 1991)

• Cells of the salivary glands

• Oral microorganisms

• Polymorphonuclear leukocytes

• Epithelial cells

• GCF

VARIOUS ENZYME BIOMARKERS IN SALIVA

1. Nakamura and slots (1983) have investigated salivary enzyme activity in chronic periodontitis, aggressive periodontitis and healthy patients. The results of their study showed increased activity of alkaline phosphatase, esterase, betaglucoronidase,and other aminopeptidases in chronic periodontitis compared to control. Their study results also showed elevated levels of butyrate esterase and cysteine aminopeptidase in aggressive periodontitis patients

2. Uitto et al (1990) showed that periodontitis patients saliva had higher levels of collagenase than control subjects

• Makela et al(1994), have demonstrated higher levels of

gelatinase activity in saliva of chronic periodontitis patients

compared to control

• Hakayawa et al(1994), revealed that total levels of TIMP 1 were

lower in chronic periodontitis subjects compared to control

• Markanen et al ( 1986) showed significantly lowered levels of

lysozyme in periodontitis patients compared to healthy subjects

• Over et al( 1993), have shown elevated levels of

myeloperoxidase in saliva of chronic and aggressive

periodontitis patients compared to control

• Nieminen et al ( 1993) in an experiment on 25 chronic

periodontitis and 25 healthy subjects showed that periodontal

treatment in the diseased subjects lowered the total enzyme

levels. Additionally they postulated that the elastase activity

in saliva correlated significantly with the number of deep

periodontal pockets and the percentage of bleeding sites. the

authors have revealed that the total enzyme activity in saliva

reflects the severity of periodontal disease

• With regard to the salivary enzyme markers in aggressive

periodontitis patients, peroxidase levels have been found to

be significantly lower in whole and parotid saliva of patients

compared to age and gender matched controls. ( Saxen et al ,

1990)

• Picareli et al(1996) have shown elevated levels of kallikerin,

the enzyme involved in conversion of high molecular weight

kininogens to bradykinin in saliva of chronic periodontitis

patients compared to control

IMMUNOGLOBULINS IN SALIVA

• Ig A• Ig G• IgM• sIg A

IMMUNOGLOBULINS IN SALIVA( SOURCES)

• The predominant immunoglobulin in saliva is Ig A. it is derived from

plasma cells in the salivary glands.

• Parotid gland derived Ig A is the predominant immune defence

mechanism in the saliva( Bienenstock et al, 1980, Nair et al , 1986)

• There are 2 isoforms A1 and A2. A2 isoform predominates in the

saliva.

• Ig G in saliva is derived from serum and GCF and generally tends to

increase in inflammation of the periodontal tissues when GCF flow

also increases

IMMUNOGLOBULINS AS SALIVARY BIOMARKERS

• Guven et al 1982, demonstrated that in whole saliva collected from gingivitis and chronic periodontitis patients, elevated Ig A levels were seen compared to healthy individuals.

• Sandholm et al 1984, evaluated levels of Ig A , IgG and IgM, in juvenile periodontitis subjects and healthy siblings and age matched controls. Their study results showed elevated levels of the mentioned immunoglobulins in the diseased group compared to health.

• Reiff et al 1984, have shown that following initial periodontal therapy including scaling and oral hygeine , the levels of Ig A dropped showing that Immunoglobulin levels in saliva is an indicator of the severity of periodontal inflammation

• Basu et al 1986, have shown elevated Ig G levels in saliva of

periodontitis patients. Following initial therapy, they found

drop in Ig G while Ig A levels increased showing that in health ,

Ig A is a protective mechanism

• In a study by Anil et al (1995), elevated levels of salivary Ig G

and Ig A were demonstrated in saliva of NIDDM Patients with

periodontitis compared to control showing that elevated

immunoglobulin levels can be regarded as a biomarkers to

describe the severity of periodontal inflammation

SPECIFIC IMMUNOGLOBULINS IN SALIVA AS BIOMARKERS

• In this context , specific immunoglobulins means, antibodies directed against the specific periodontopathic bacteria

• Mansheim et al 1980, evaluated IgA levels against Bacteroides asaccharolyticus ( P.gingivalis) in 8 patients with rapidly progressing periodontitis and chronic periodontitis. Their study results showed that in response to infection from the subgingival biofilm, there is a concurrent rise in antibody titres to P.gingivalis

• In another study, Sandholm et al( 1987), showed raised Ig G levels directed to A.a in the serum and saliva of aggressive periodontitis patients compared to healthy control

• Nieminen et al (1993) in a study reported that the concentration of specific Ig G and Ig A antibodies to Aa in saliva of patients with advanced periodontitis correlated significantly with corresponding antibody titers in the serum of those patients. It is concluded that for severe aggressive periodontitis , saliva samples could be used diagnostically to assess the serum antibody response to Aa

PROTEIN BIOMARKERS IN SALIVA USEFUL FOR PERIODONTAL DIAGNOSIS

• Cystatins

• Epidermal growth factor

• Fibronectin

• Lactoferrin

• Platelet activating factor

• Vascular endothelial growth factor

FIBRONECTIN• This section highlights the other proteins other than enzymes

and immunoglobulins that can be used as biomarkers• Fibronectin is an important protein in periodontal

homeostasis. It is a glycoprotein that mediated adhesion between cells and is also involved in chemotaxis, inflammation, migration and wound healing. In a study by Lamberts et al ( 1989), that assessed fibronectin levels in saliva of periodontitis patients and healthy subjects, it was found that there was elevation of fibronectin in saliva of the diseased group compared to control

CYSTATINS• Cysteine proteinases are proteolytic enzymes originating from

pathogenic bacteria, inflammatory cells, osteoclastic cells and fibroblasts. these enzymes have collagenolytic activity which may cause tissue destruction( Cutler et al , 1995).

• Cystatins are inhibitors of cysteine proteinases and may function in modulating enzyme activity in the periodontium. Cystatins are present in a variety of body fluids such as blood, saliva and GCF.

• Cystatins found in saliva are believed to originate from the submandibular gland and to a lesser extent from the parotid gland.

• Blankenvoorde et al( 1997) analysed cystatin levels in GCF and saliva of chronic periodontitis patients. Their study results showed that Cystatin C,S and SN were found in saliva and not in GCF where only Cystatin A was found.The increased cystatin activity was demonstrated in periodontitis patients compared to control.

• Henskens et al (1996) in their study showed that total cystatin activity in saliva is an indicator of periodontal disease severity .Morover , initial and surgical periodontal therapy resulted in a drop in cystatin levels in saliva in the periodontitis group compared tp control

• From various studies , researchers have hypothesised that salivary glands respond to periodontitis by enhanced synthesis of acinar proteins. Also it has been documented that the parotid saliva is the major source of Cystatin

PLATELET ACTIVATING FACTOR (PAF)

• Platelet activating factor is an important mediator of inflammation

• It is a phospholipid mediator which has proinflammatory and procoagulant effects.

• Garito et al (1995) in their study on whole saliva from 69 periodontitis and healthy subjects demonstrated statistically higher levels of PAF in the periodontitis group. PAF levels in saliva correlate positively with % of sites with probing depth greater than 4 mm and bleeding.

• Rasch et al (1995) in their longituidnal study found that there was a statistically significant reduction in salivary PAF levels in periodontitis patients following scaling and oral hygeine regimen

EPIDERMAL GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR

• Epidermal growth factor (EGF) is involved in oral wound healing and functions with hormone like properties. In humans, parotid gland is the active source of EGF (Thesleff et al, 1988)

• Elevated salivary EGF levels have been shown in juvenile periodontitis patients compared to control by Hormia et al (1993).

• Oxford et al (1998) have shown that periodontal surgery increases the levels of EGF in saliva

• Vascular endothelial growth factor (VEGF) is also known as

vascular permeability factot or vasculotropin is a

multifunctional angiogenic cytokine important in

inflammation and wound healing

• This growth factor is an important component of whole saliva(

Taichman et al, 1998). Higher levels of VEGF have been shown

in saliva of chronic periodontitis patients by Booth et al (1998)

FREE AMINO ACIDS

• Free amino acids in saliva are believed to be derived from

bacterial metabolism and tissue degradation

• Syrjanen et al ( 1984,1987) in 2 studies have shown that

salivary proline levels were elevated in periodontitis patients

compared to control

EPITHELIAL KERATINS AS SALIVARY BIOMARKERS IN PERIODONTAL DISEASES

• Epithelial cells from the lining mucosa of the oral cavity are

found in saliva

• To study epithelial cell function in periodontal disease and

diagnosis, specific keratin antigens need to be evaluated.

• Mc laughlin et al (1996) have shown elevated GCF keratin

levels in periodontitis patients compared to control. Such a

trend was not seen with saliva

INFLAMMATORY CELLS AS SALIVARY BIOMARKERS

• The number of leukocytes in saliva vary from person to person and cell counts vary even during different times of the day

• The majority of salivary leukocytes enter the oral cavity through the gingival crevice( Schiott and Loe, 1970)

• Studies in 1960s and 1970s by Klinkhammer described orogranulocyte migratory rate (OMR).

• The OMR was found to be elevated in gingival and periodontal disease and correlates with the gingival index

• In another study Raeste et al(1978) showed that in mouthrinse samples of periodontitis patients , the total leukocyte count correlated with the severity of periodontal inflammation

OCCULT BLOOD AS A SALIVARY BIOMARKER IN PERIODONTAL DISEASE

• The presence of Occult blood in saliva has been related to

gingival inflammation

• This has been examined by Kopczyk et al 1995, as a home

screening test

• According to the authors, sensitivity of 75.9 % and specificity

of 90.5% has been observed for this home based test

SALIVARY IONS AS BIOMARKERS OF PERIODONTAL DISEASE

• Calcium is the ion that has been most intensely studied as a

potential marker for periodontal disease in saliva

• Sewon et al ( 1990) have shown that periodontitis patients

have high salivary calcium to phosphate ratio

• In another study, the same authors showed higher levels of

salivary calcium in 20 periodontitis patients compared to 15

control.

HORMONES AS SALIVARY BIOMARKERS IN PERIODONTAL DISEASE

• The main hormones that are used as salivary biomarkers are cortisol and melatonin

• Recent studies have suggested that emotional stress is a risk factor for periodontal disease( Breivik et al,1996, Genco et al ,1996)

• The presence of cortisol in saliva has been described as early as in 1959 by Shannon et al.

• High salivary cortisol levels have been demonstrated in severe periodontitis patients , a high level of financial strain, low levels of coping to stress

• Melatonin is N acetyl 5 methoxy tryptamine.it is a pineal gland hormone that is secreted by the pinealocytes. The extra pineal sources of melatonin are the ovaries, testis, retina , gut epithelium and brain cells

• Salivary melatonin depicts the fraction that is not protein bound. Laakso et al (1993) have shown that 33% of the total body melatonin is found in saliva. morover in saliva, melatonin is an immunomodulator, antioxidant and oncostatic agent .

• Antonio Cutando (2006) in their study showed that melatonin levels in saliva of periodontitis patients correlated inversely with the CPI score. In another study the same authors have shown lowered salivary melatonin levels in diabetic subjects compared to control.

BACTERIA AS SALIVARY BIOMARKERS• Specific species of bacteria colonizing the subgingival

environment have been implicated in the pathogenesis of periodontal disease. It has been suggested that microorganisms from dental plaque can survive in saliva and can utilise salivary components as substrates for growth and survival.

• Asikainen et al in their study on Aggressive periodontitis patients showed that there was equally effective recovery of Aa from subgingival plaque, stimulated and unstimulated saliva.

• Umeda et al (1998), have shown that periodontpathic bacteria survive in saliva and subgingival plaque of periodontitis patients. Using the PCR technique, a fair aggrement was seen between the presence of Pg, Bf and Td in saliva and subgingival plaque samples

• Salivary levels of microbes have been found to vary with

periodontal status and treatment measures. Danser et al(1996)

have shown significant reduction in the counts of

periodontopathic bacteria in saliva followin scaling and root

planing and periodontal surgery

• An oral microbial rinse test also called ORATEST has been

described by Rosenberg et al( 1989). In this study the ORATEST

has been described as a simple test to estimate microbes in

saliva

VOLATILE SULPHUR COMPOUNDS AS SALIVARY BIOMARKERS

• These are predominantly hydrogen sulfide, dimethyl disulfide, methyl mercaptan, skatole

• These compounds have been associated with oral malodor( Rosenberg and McCulloch, 1992).

• Salivary volatiles have been described as markers of periodontal disease severity

• Kostlec et al( 1980,81) have shown that elevation in salivary picolines and pyridines are associated with periodontal destruction.

• Kozlovsky et al (1994), have shown a significant assocoation between salivary levels of vsc and BANA scores

CYTOKINES AND ACUTE PHASE PROTEINS AS SALIVARY BIOMARKERS

• Cytokines are protein molecules that are implicated in

inflammation ,wound healing and the immune response

• Cytokines may be proinflammatory or antiinflammatory.

• Miller CS et al have shown elevated levels of IL 1 beta and

MMP8 in saliva of periodontitis subjects

• Christodoulides et al , 2007 have shown elevated IL 1 alpha

and beta levels in whole unstimulated saliva in periodontitis

patients compared to healthy control

• Frodge BD et al ,2008 have shown that elevated YNF alpha

levels in saliva correlates with the clinical indicators of

periodontal disease

• Pederson et al ,1998 have shown elevated levels of the acute

phase proteins, alpha 2 macroglobulin, alpha 1 antitrypsin, C

reactive protein in saliva of periodontitis patients compared to

healthy control.

OXIDATIVE STRESS MOLECULES AS BIOMARKERS IN SALIVA

• There is ample evidence to highlight the role of ROS in the pathogenesis of periodontal disease.

• Panjamoorthy et al (2005) have shown elevated levels of thibarbaturic acid reactive substances in the saliva of chronic periodontitis

• Sawamoto et al (2005), have shown elevated 8 OHDG in saliva of periodontitis patients compared to control

• In a successive experiment, the same authors have shown that elevated 8OHDG levels in saliva correlate with the presence of P.gingivalis in subgingival plaque

CHALLENGES IN SALIVARY BIOMARKER APPLICATIONS

• Saliva has been known to be of diagnostic value for almost

2000 years

• Its value has been exploited only recently with the advent of

genomic and proteomic technologies

• It can be easily sampled in a private practice and applied on to

a chair side assay kit

WHAT ARE THE ASSAY TECHNIQUES FOR THE SALIVARY BIOMARKERS

• PROTEIN EXTRACTION FROM WHOLE SALIVA

• ELECTROPHORETIC TECHNIQUES

• ELISA

• MICROARRAY TECHNOLOGY ( LAB ON A CHIP)

• PCR AND GENOMIC TECHNOLOGY

• MICROBIAL CULTURE TECHNIQUES

VISION AND CHALLENGES IN DEVELOPING AND IMPLEMENTING SALIVARY BIOMARKERS

• The missing tools from the diagnostic tool box are biomarkers

that predict the onset of periodontal disease ,its progression

and prognostic relevance

• The NIDCR has instituted seven research awards for

developing microfliuidic and microelectromechanical systems

for saliva research .

• Barnfather et al have investigated the effect of immediate

feedback from a point of care test evaluating the nicotine

metabolites in saliva in connection with a smoking cessation

programme. They found that good results could be obtained

when patients were educated with a chair side test

RECENT ADVANCES IN SALIVRY BIOMARKER BIOLOGY

• Herr et al have developed a clinical point of care diagnostic

test that uses a monolithic cartridge based system to quickly

measure MMP 8 levels in saliva in a hands free manner.

• Successively, the same group has developed a portable

diagnostic device called the “integrated microfluidic platform

for oral diagnostics” . This is a compact hand held lab on chip

that rapidly detects CRP, IL 1 AND MMP 8 in saliva samples

HUMAN SALIVARY PROTEOME AND TRANSCRIPTOME PROJECT

• Based on NIDCR FUNDING

• By using electrophoretic and shotgun proteomic techniques,

salivary proteins have been isolated and fractioned

• 1166 salivary proteins have been identified

• 914 from parotid saliva

• 917 from submandibular and sublingual saliva

• The university of California at LA has devised a database known as

Salivary proteome knowledge

WHAT IS IN THE FUTURE

SALIVA THE ELIXIR OF

DIAGNOSIS

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