Dr Rupali's Online Clinic: Saliva

INTRODUCTION

Saliva is a most valuable oral fluid that often is taken for granted. It is critical to the preservation and maintenance of oral health, yet it receives little attention until quantity or quality is diminished.

Saliva is a complex fluid, produced by the salivary glands. It is a heterogeneous fluid comprising proteins, electrolytes, small organic molecules & compounds transported from the blood, constantly bathes the teeth & oral mucosa.

Saliva is one of the most important fluids in the human body. Its status in the oral cavity is at par with that of blood i.e. to remove waste , supply nutrients and protection of cells.

It acts as a cleansing solution, an ion reservoir, a lubricant & a buffer.

In addition to its other host - protective properties, saliva could constitute a first line of defense against free radical-mediated oxidative stress, since the process of mastication & digestion of ingested foods promotes a variety of reactions, including lipid peroxidation.

Individuals with a deficiency of salivary secretion experience difficulty in eating, speaking, swallowing & become prone to mucosal infections & rampant caries.

Saliva is composed of more than 99% water and less than 1% solids , mostly electrolytes and proteins, the latter giving saliva its characteristic viscosity. Normally the daily production of whole saliva ranges from 0.5 to 1.0 litres.

90% of the whole saliva is produced by three paired major salivary glands

Parotid Gland

Submandibular gland

Sublingual gland

CLASSIFICATION

1.Based on anatomic location

· Parotid gland

· Sub mandibular gland

· Sub lingual gland

· Accessory glands (labial, lingual, palatal buccal,glossopalatine and retromolar)

2. Based on size and amount of secretion

· Major salivary glands

· Minor salivary glands

3. Based on type of secretion

· Serous

· Mucous

· Mixed

Parotid glands - Purely serous

Submandibular-Predominantly serous, Mixed

Sublingual - Predominantly mucous , Mixed

Labial,Buccal,Lingual{Ant.}- Predom. Mucous , Mixed

Palatine,Glossopalatine - Purely mucous.

Posterior part of the tongue - Purely mucous

Von Ebner’s Glands - Purely serous

COMPOSITION

Saliva is composed of a variety of electrolytes, including sodium, potassium, calcium, magnesium, bicarbonate, and phosphates.

Also found in saliva are immunoglobulins, proteins, enzymes, mucins, and nitrogenous products, such as urea and ammonia.

These components interact in related function in the following general areas:

(1) bicarbonates, phosphates, and urea act to modulate pH and the buffering capacity of saliva; (2) macromolecule proteins and mucins serve to cleanse, aggregate, and/or attach oral microorganisms and contribute to dental plaque metabolism;

(3) calcium, phosphate, and proteins work together as an antisolubility factor and modulate demineralization and remineralization;

(4) immunoglobulins, proteins, and enzymes provide antibacterial action.

Salivary components, particularly proteins, are

multifunctional (performing more than 1 function)

redundant (performing similar functions but to different extents)

amphifunctional (acting both for and against the host).

Saliva is a very dilute fluid, composed of more than 99% water.

Saliva is not considered an ultrafiltrate of plasma.

Initially, saliva is isotonic, as it is formed in the acini, but it becomes hypotonic as it travels through the duct network.

The hypotonicity of unstimulated saliva

· allows the taste buds to perceive different tastes without being masked by normal plasma sodium levels.

· Hypotonicity, especially during low flow periods, also allows for expansion and hydration of mucin glycoproteins, which protectively blanket tissues of the mouth.

· Lower levels of glucose, bicarbonate, and urea in unstimulated saliva augment the hypotonic environment to enhance taste.

The normal pH of saliva is 6 to 7, meaning that it is slightly acidic. The pH in salivary flow can range from 5.3 (low flow) to 7.8 (peak flow).

Major salivary glands contribute most of the secretion volume and electrolyte content to saliva, whereas minor salivary glands contribute little secretion volume and most of the blood-group substances.

FUNCTIONS

PROPERTIES OF SALIVA

• Consistency : Slightly cloudy

• Reaction : Usually slightly acidic

• PH : 5-8

• Specific gravity : 1.0024 – 1.0061

• Freezing point :0.07 – 0.34 degree Celsius

• Osmotic pressure : ( 700-1000m osmol/litre )

TIME OF ORIGIN

STAGES OF DEVELOPMENT

• STAGE I-Bud formation:

1. Induction of proliferation of oral epithelium by underlying mesenchyme.

2. Individual salivary glands arise as a proliferation of oral epithelial cells, forming a focal thickening that grows into the underlying ectomesenchyme.

3. These long epithelial cords undergo repeated dichotomous branching, called, “Branching morphogenesis”, that produces successive generations of buds & a hierarchial ramification of the gland & the mesenchymal cells condense around the bud.

· STAGE II:Formation and growth of epithelial cord

· STAGE III: Initiation of branching in terminal parts of epithelial cord and continuation of glandular differentiation.

· STAGEIV: Dichotomous branching of epithelial cord and lobule formation.

1. The development of a lumen within the branched generally occurs first in the distal end of the main cord & finally in the central portion of the main cord.

2. The lumen form within the ducts before they develop within the terminal buds.

3. Some studies have suggested that lumen formation may involve the apoptosis of centrally located cells in the cords.

· STAGE V:Canalization of presumptive ducts.

1. Following development of the lumen in the terminal buds, the epithelium consists of two layers of cells.

a) The cells of the inner layer eventually differentiate into the secretory cells of the mature gland, mucous / serous.

b) Some cells of the outer layer form the contractile myoepithelial cells that are present around the secretory end pieces & intercalated ducts.

2. As the epithelial parenchymal components increase in size & number, the associated mesenchyme ( connective tissue ) is diminished, although a thin layer of connective tissue remains, surrounding each secretory end piece & duct of the adult gland.

· STAGE VI: Cytodifferentiation.

Cells of ® Terminal tubule cell ® Proacinar cells®Acinar cells

Bulb region ¯

Intercalated duct cell

• Septa ( Thicker partitions of the connective tissue ) which are continuous with the connective tissue capsule surrounding the gland parenchyma into lobes & lobules & carry the blood vessels & nerves that supply the parenchymal components & the excretoryducts.

The cells of the secretory end pieces & ducts attain maturity during the last 2 months of gestation.

The glands continue to grow post natally with the volume proportion of acinar tissue increasing & the volume proportions of ducts connective tissue & vascular elements decreasing upto 2 years of age.

MORPHOLOGIC CHARACTERISTICS OF MAJOR SALIVARY GLANDS

Parotid gland

Ø Largest of all the salivary glands

Ø Purely serous gland that produce thin , watery amylase rich saliva

Ø Superficial portion lies in front of external ear & deeper portion lies behind the ramus of mandible

Ø Stensen's Duct (Parotid Papilla) opens out adjacent to maxillary second molar.

Submandibular Gland

Ø Second largest salivary gland

Ø Mixed gland

Ø Located in the posterior part of floor of mouth,adjacent to medial aspect of mandible & wrapping around the posterior border of mylohyoid muscle.

Ø Wharton's Duct opens beneath the tongue at sub-lingual caruncle lateral to the lingual frenum

Sublingual Gland

Ø Smallest salivary gland

Ø Mixed gland but mucous secretory cells predominate.

Ø Located in anterior part of floor of mouth between the mucosa and mylohyoid muscle

Ø Opens through series of small ducts (ducts of rivinus) opening along the sub-lingual fold & often through a larger duct (bartholin’s duct)

The minor salivary glands:

Ø Estimated numbers is 600-1000.

Ø Exist as small,discrete,aggregates of secretory tissue present in the submucosa through out most of the oral cavity, except the gingival & anterior part of the hard palate.

Ø Predominantly mucous glands,except for Von Ebners glands(purely serous)

Ø Here intercalated & striated ducts are poorly developed.

VASCULAR SUPPLY

PAROTID GLAND

Arterial: Ext.Carotid Artery and its branches

Venous: Ext.Jugular Vein

Lymphatic: Parotid Nodes® Upper deep cervical nodes

SUBMANDIBULAR GLAND

Arterial: Facial Artery , Lingual Artery

Venous: Common Facial Vein /Lingual Vein

Lymphatic: Submandibular Lymph nodes

SUBLINGUAL GLAND

Arterial: Lingual and Submental Arteries

Venous: Lingual Vein

INFLUENCE OF BLOOD SUPPLY ON SALIVARY SECRETION

q Extensive blood supply is required for rapid salivary secretion.

q Salivation indirectly dilates blood vessels providing increased nutrition.

q Large increase in blood flow accompanies salivary secretion.

INNERVATION

Parasympathetic innervation to major salivary glands

} Otic ganglion suplies the parotid gland.

} Submandibular ganglion supplies the other major glands.

Sympathetic innervation

Promotes the flow of saliva and stimulates muscle contraction at salivary ducts

REGULATION OF SALIVARY SECRETION

The secretion of saliva is controlled by a salivary center composed of nuclei in the medulla.

Three types of triggers, or stimuli, for this production are

· mechanical (the act of chewing),

· gustatory (with acid the most stimulating trigger and sweet the least stimulating),

· olfactory (a surprisingly poor stimulus).

Other factors affecting secretion include

· psychic factors such as pain,

· certain types of medication

· various local or systemic diseases affecting the glands themselves.

Salivary glands are innervated by both sympathetic and parasympathetic nerve fibers.

· When sympathetic innervations dominate, the secretions contain more protein from acinar cells,

· parasympathetic innervations produce a more watery secretion.

SALIVARY GLAND STRUCTURE

Composed of parenchymal elements supported by connective tissue

The types of cells found in the salivary glands are duct system cells, acinar cells, and myoepithelial cells.

• Intercalated duct : main duct connecting acinar secretions to rest of the gland, not involved in modification of electrolytes

• Striated duct: electrolyte regulation in resorbing sodium

• Excretory duct: continuing sodium resorption and secreting potassium

• Inter cellular canaliculi : These are the extensions of the lumen of the end piece between adjacent secretory cells that serve to increase the terminal surface area available for secretion.

• Secretory end pieces: branched ducts, terminating in spherical or tubular secretory end pieces/ acini.

Secretory cells: There are two types of secretory cells.

1.serous cells

2.mucous cells

These two cells differ in structure, as seen in classical histologic & electron microscopic analyses, & in the types of macromolecular components that they produce & secrete.

1.SEROUS CELLS:

a) These are spherical, consisting of 8-12 cells surrounding a central lumen.

b) Cells are pyramidal with a broad base adjacent to the connective tissue stroma & a narrow apex forming part of the lumen of the end piece.

c) The lumen usually has finger like extensions located between adjacent cells called inter cellular canaliculi.

d) Spherical nuclei are located basally, occasionally binucleated cells are seen.

e) Secretory granules in which macromolecular components of saliva are stored, are present in the apical cytoplasm.

f) These cells are joined by intercellular junctions.

a.Zonula occludens( tight junction)

b.Zonula adherens(Adhering junction)

c.Macula adherens(desmosome)

Tight junctions exhibit a selective permeability, allowing the passage of certain ions & water.

These cells are attached to the basal lamina & the underlying connective tissue by hemidesmosomes.

2.MUCOUS ACINI:

a) These have a tubular configuration.

b) When cut in cross section, these tubules appear as round profiles with mucous cells surrounding a central lumen of larger size than that of serous end pieces

c) Mucous end pieces in the major salivary glands & some minor salivary glands have serous cells associated with them in the form of a demilune or cresent covering the mucous cells at the end of the tubule.\

d) The most prominent feature of the mucous cell is the accumulation in the apical cytoplasm of large amounts of secretory product (mucus), which compresses the nucleus & endoplasmic reticulum & golgi complex against the basal cell membrane.]

e) Unlike serous cells, however, mucous cells lack intercellular canaliculi, except for those covered by demilune cells.

3.MYOEPITHELIAL CELLS:

a)These are basket shaped cells, contractile in nature & are associated with the secretory end pieces & intercalated ducts of the salivary glands.

b) They are located between the basal lamina & the secretory/duct cells & are joined to the cells by desmosomes.

c) These are similar to the smooth muscle cells but are derived from the epithelium.

d) Contraction of the myoepithelial cells is due to actin & myosin, which helps to provide support for the end pieces during active secretion of saliva.

e) These may help to expel the primary saliva from the endpiece into the duct system.

f) They provide signals to the acinar secretory cells that are necessary for maintaining cell polarity & the structural organization of the secretory end piece.

g) They produce a no. of proteins that have tumour suppressor activity, such as proteinase inhibitors ( ex : tissue inhibitor of metalloproteinases ) & antiangiogenesis factors

h) provide a barrier against invasive epithelial neoplasms.

FORMATION OF SALIVA

The salivary glands are composed of specialized epithelial cells, and their structure can be divided into two specific regions: the acinar and ductal regions.

The acinar region is where fluid is generated and most of the protein synthesis and secretion takes place. Amino acids enter the acinar cells by means of active transport, and after intracellular protein synthesis, the majority of proteins are stored in storage granules that are released in response to secretory stimulation (Young and Van Lennep, 1978; Castle, 1993).

Three models have been described for acinar fluid secretion

1. active transport of anions into the lumen and passage of water according to the osmotic gradient from the interstitial fluid into the salivary lumen (Turner et al., 1993).

2. The initial fluid is isotonic in nature and is derived from the local vasculature.

3. While acinar cells are water-permeable, ductal cells are not.

Ductal cells actively absorb most of the Na+ and Cl- ions from the primary salivary secretion and secrete small amounts of K+ and HCO3 - and some proteins.

The primary salivary secretion is thus modified, and the final salivary secretion as it enters the oral cavity is hypotonic (Baum, 1993).

The autonomic nervous system (sympathetic and parasympathetic) controls the salivary secretion. The signaling mechanism involves the binding of neurotransmitter (primarily acetylcholine and norepinephrine) to plasma membrane receptors and signal transduction via guanine nucleotide-binding regulatory proteins (G-proteins) and activation of intracellular calcium signaling mechanisms (for reviews, see Baum, 1987, 1993; Ambudkar, 2000).

SALIVARY GLAND SECRETIONS

Saliva can be considered as gland-specific saliva and whole saliva.

Gland-specific saliva can be collected directly from individual salivary glands: parotid, submandibular, sublingual, and minor salivary glands. Useful for the detection of gland-specific pathology, i.e., infection and obstruction.

Whole saliva (mixed saliva) is a mixture of oral fluids and includes secretions from both the major and minor salivary glands, in addition to several constituents of non-salivary origin, such as gingival crevicular fluid (GCF), expectorated bronchial and nasal secretions, serum and blood derivatives from oral wounds, bacteria and bacterial products, viruses and fungi, desquamated epithelial cells, other cellular components, and food debris. Used when salivary analysis is used for the evaluation of systemic disorders.

Saliva can be collected with or without stimulation.

Stimulated saliva is collected by masticatory action (i.e., from a subject chewing on paraffin) or by gustatory stimulation (i.e., application of citric acid on the subject's tongue; Mandel, 1993).

Unstimulated saliva is collected without exogenous gustatory, masticatory, or mechanical stimulation.

COLLECTION OF SALIVA

• Non invasive, non painful techniques exist to collect whole saliva, as well as saliva from the individual major & minor salivary glands .

• Whole saliva is easily obtained & is in most case a good indicator of whole mouth dryness.

• Diseases of salivary gland can often be diagnosed from the secretions obtained directly.

• The quantification of salivary output is referred to as sialometry.

University of Southern California School of Dentistry guidelines

• Unstimulated whole saliva collection always should precede stimulated whole saliva collection.

• The patient is advised to refrain from intake of any food or beverage (water exempted) one hour before the test session.

• Smoking, chewing gum and intake of coffee also are prohibited during this hour.

• The subject is advised to rinse his or her mouth several times with distilled water and then to relax for five minutes.

• Keep his mouth slightly open and allow saliva to drain into the tube.

• Should last for five minutes

Collection Of Stimulated Saliva

• Paraffin method (Masticatory stimulus ): Ask the patient to hold a piece of paraffin in his or her mouth until it becomes soft ( about 30 seconds) & then to swallow the saliva which has collect in his mouth. Now the patient is asked to chew the piece of wax in his usual manner of chewing for exactly 2 minutes & then to expectorate the accumulated saliva into the receiving vessel .The volume of saliva is read off the vessel & flow is expressed as ml/min.

• Citric Acid method ( Gustatory Stimulus ): A 2% solution of citric acid is swabbed on to the laterodorsal surface of the tongue every 30 seconds for a period of 2 min & then saliva is expectorated into the receiving vessel. As in the paraffin method, the whole procedure is repeated twice more, for a total time of 6 mins. As before, the flow is expressed as ml/min.

SALIVARY FLOW RATE

There is great variability in individual salivary flow rates.

The accepted range of normal flow for unstimulated saliva is anything above 0.1 mL/min.

For stimulated saliva, the minimum volume for the accepted norm increases to 0.2 mL/min.

Any unstimulated flow rate below 0.1 mL/min is considered hypofunction.

In a 1992 study, the critical range separating persons with normal gland function from those with hypofunction was more precisely identified as unstimulated whole salivary flow rates between 0.12 and 0.16 mL/min.

If individualized base rates have been established, then a 50% reduction in flow should be considered hypofunction.

On average, unstimulated flow rate is 0.3 mL/min

Salivary flow during sleep is nearly zero.

Stimulated flow rate is, at maximum, 7 mL/min.

Stimulated saliva is reported to contribute as much as 80% to 90% of the average daily salivary production.

FACTORS AFFECTING SALIVARY FLOW RATE

Diurnal variation:

• Protein concentrations tend to be high in the afternoon.

• Sodium & chloride concentrations are high in the morning, while potassium is high in the early afternoon.

• The calcium concentration increase at night.

Duration of stimulus:

• If the salivary glands are stimulated for long than 3 minutes, the concentration of many components is reduced.

• Chloride concentrations fall during periods of stimulation.

Hormonal Influences

• Aldosterone: It results in increased sodium reabsorption in the striated ducts.

• Antidiuretic hormone (ADH): Stimulates water reabsorption by the striated duct cells.

• Other hormones: Thyroxine results in increase salivary secretion

• Local hormones: Bradykinin & its predecessor kallidin, result in increased salivary secretion.

ANOMALIES

I.Developmental

Aberrant Salivary Glands

Aplasia and Hyperplasia

Atresia

II.Obstructive conditions

Sialolithiasis

Mucocele

Necrotizing Sialometaplasia

III. Inflammatory Diseases

Viral- Mumps , H.I.V. Associated

Bacterial - Sialadenitis

IV.Neoplastic Diseases

Benign

Malignant

V.Degenerative Conditions

Sjogren’s Syndrome

Ionizing Radiation

VI.Xerostomia

XEROSTOMIA

• It is a condition of reduced or absent salivary flow,leading to the dryness of the mouth.

• It is not a disease by itself, but a symptom associated with alterations of salivary function.

• The principal causes of salivary gland hypofunction & xerostomia

Oral symptoms

1. Dry mouth ( xerostomia )

2. Often thirsty

3.Dysphagia (difficulty with swallowing )

4. Dysphonia ( difficulty with speaking )

5. Dysgeusia ( abnormal taste sensation )

6. Difficulty with eating dry foods

7. Need to frequently sip water while eating

8. Difficulty with wearing dentures

9. Often do things to keep mouth moist

10.Burning, tingling,sensation on the tongue.

11.Fissures & sores at corners of lips

Clinical signs

1. Dryness of lining oral tissues

2. Loss of glistening of the oral mucosa

3. Dryness of the oral mucous membranes

4. Oral mucosa appears thin & pale

5. Tongue blade/mirror/a gloved finger may adhere to the soft tissues

6. Fissuring & lobulation of the dorsum of the tongue & lips

7. Angular cheilitis

8. Candidiasis on tongue & palate

9. Increased incidence of dental caries

10. Thicker, more stringy saliva

11. Swelling of glands

12. Increase in inflammatory gingival diseases

13. Rapid tooth destruction associated with cervical or cemental caries

Treatment of salivary hypofunction & xerostomia :

• Systemic Therapy:

Bromohexine, anethole, triothiline & pilocarpine Hcl all three should be used under the care of a specialist & following medical examination.

• Local Therapy

SALIVARY SUBSTITUTES

Carboxy methyl cellulose (CMC) based

l Imparts lubrication and viscosity

l Sorbitol or xylitol are added to provide surface activity and as a sweetner.

l Have surface tension greater than natural saliva.

Mucin based

Animal mucins derived from procine gastric tissues / bovine salivary glands.

Salts are addeded to mimic the electrolyte content of natural saliva

HYPERSALIVATION

• It is also known as sialorrhea, ptyalism.

• It may lead problems in oral motor coordination, including reduced muscle tone around the mouth & a reduced ability to swallow.

• Causes:

1. After extensive surgery for oral or oropharyngeal disorders.

2. As a result of stomatitis, psychological factors, & the use of some drugs, Ex: benzodiazepines,captopril

3. Treatment

i) Drugs – anticholinergics.

ii) Surgical – depending on the nature of the anomaly.

DIGNOSTIC APPLICATIONS

The most commonly used laboratory diagnostic procedures involve the analyses of the cellular and chemical constituents of blood.

There are several ways by which serum constituents that are not part of the normal salivary constituents (i.e., drugs and hormones) can reach saliva.

Within the salivary glands, transfer mechanisms include intracellular and extracellular routes.

The most common intracellular route is passive diffusion, although active transport has also been reported.

Ultrafiltration, which occurs through the tight junctions between the cells, is the most common extracellular route (Drobitch and Svensson, 1992; Haeckel and Hanecke, 1993; Jusko and Milsap, 1993).

Serum molecule reaching saliva by diffusion must cross five barriers: the capillary wall, interstitial space, basal cell membrane of the acinus cell or duct cell, cytoplasm of the acinus or duct cell, and the luminal cell membrane (Haeckel and Hanecke, 1996; Fig. 2).

Serum constituents are also found in whole saliva as a result of GCF outflow. Depending on the degree of inflammation in the gingiva, GCF is either a serum transudate or, more commonly, an inflammatory exudate that contains serum constituents.

Advantages

1. saliva can be collected without breaking the skin or entering the body in any other way, it has obvious advantages for multiple noninvasive collections and for obtaining samples from those whom, for cultural reasons or age or because of physical or mental handicaps, it would be unethical to collect blood samples.

2. saliva levels are a more accurate reflection of the active hormone in the body, especially for steroid hormones, which are strongly bound in blood by specific binding globulins (Read 1989).

3. Saliva can be collected with devices so that it will be stable at room temperature for extended periods.

4. Many of the hazards associated with blood collection do not apply to saliva. There is no need for sharps, which have the potential for cross contamination among patients when used improperly and present a danger to health care personnel.

5. Because of the low concentrations of antigens in saliva, HIV and hepatitis infections are much less of a danger from saliva than from blood (Major et al. 1991).

6. Because of diurnal and monthly variations, several steroid hormones need multiple samples collected early in the morning or late at night or at the same time every day for a month to give meaningful results (Read 1989). Such collections are often very expensive, inconvenient or impossible to do with blood.

7. Nonpolar analytes are released into saliva through the membranes via a mechanism that is not flow dependent. Therefore, concentrations remain constant relative to blood levels with stimulated and unstimulated collections (Vining et al. 1983a).

8. The presence of secretory leukocyte protease inhibitor (SLPI) may be another factor contributing to the safety of saliva as a diagnostic specimen.as it expresses antiviral activity against free HIV-1 in a model

9. No special equipment is needed for collection of the fluid.

10. Diagnosis of disease via the analysis of saliva is potentially valuable for children and older adults, since collection of the fluid is associated with fewer compliance problems as compared with the collection of blood.

11. Cost-effective approach for the screening of large populations.

DISADVANTAGES

1. samples are not sterile and are subject to bacterial degradation over time.

2. Absorbing specimens on cotton may contribute interfering substances to the extract.

3. Interpretation of saliva assays is still difficult.

4. Because blood concentrations of steroid hormones are several-fold higher than saliva levels, much has been written about the problems of contamination from bleeding gums.

5. Polar hormones, such as thyroxine, and the peptide hormones are subject to variation by flow rate, so reliable levels cannot be obtained in saliva at this time (Read 1989).

6. A few kits offer saliva controls with the reagents

Saliva is used for the diagnosis

(1) Hereditary Diseases

(2) Autoimmune Diseases

(3) Malignancy

(4) Infectious Diseases

(5) Drug Monitoring

(6) The Monitoring Of Hormone Levels

(7) Diagnosis Of Oral Disease With Relevance For Systemic Diseases

Some systemic diseases affect salivary glands directly or indirectly, and may influence the quantity of saliva that is produced, as well as the composition of the fluid. These characteristic changes may contribute to the diagnosis and early detection of these diseases.

Cystic fibrosis (CF) is a genetically transmitted disease of children and young adults, which is considered a generalized exocrinopathy. CF is the most common lethal autosomal-recessive disorder in ,with an incidence of 1 in 2500 and a carrier frequency of 1 in 25-30 of the population.

The gene defect causing CF is present on chromosome 7 and codes for a transmembrane-regulating protein called the cystic fibrosis transmembrane conductance regulator (CFTR; Riordan et al., 1989; Dinwiddie, 2000).

A defective electrolyte transport in epithelial cells and viscous mucus secretions from glands and epithelia characterize this disorder (Grody, 1999).

The CFTR is also important for plasma membrane recycling (Bradbury et al., 1992).

The organs mostly affected in CF are:

· sweat glands, which produce a secretion with elevated concentrations of sodium and chloride

· lungs, which develop chronic obstructive pulmonary disease

· pancreas, resulting in pancreatic insufficiency (Davis, 1987).

Since a large number of identified mutations in the CF gene exist, DNA analysis is not used for diagnosis of the disease. The diagnosis is derived from the characteristic clinical signs and symptoms and analysis of elevated sweat chloride values. The abnormal secretions present in CF caused clinicians to explore the usefulness of saliva for the diagnosis of the disease.

· Most studies agree that saliva of CF patients contains increased calcium levels (Mandel et al., 1967; Blomfield et al., 1976; Mangos and Donnelly, 1981). Elevated levels of calcium and proteins in submandibular saliva from CF patients were found, and resulted in a calcium-protein aggregation which caused turbidity of saliva (Boat et al., 1974).

· The elevated calcium and phosphate levels in the saliva of children diagnosed with CF may explain the fact that these children demonstrate a higher occurrence of calculus as compared with healthy controls (Wotman et al., 1973).

· The submandibular saliva of CF patients was also found to contain more lipid than saliva of non-affected individuals, and the levels of neutral lipids, phospholipids, and glycolipids are elevated.

· Elevations in electrolytes (sodium, chloride, calcium, and phosphorus), urea and uric acid, and total protein were observed in the submandibuar saliva of CF patients (Mandel et al., 1967).

· Minor salivary glands are also affected.

· Elevated levels of sodium and a decrease in flow rate were reported for these glands in CF patients (Wiesman et al., 1972).

· However, the parotid saliva of CF patients does not demonstrate qualitative changes as compared with that of healthy individuals.

· Amylase and lysozyme activity in the parotid saliva of CF patients was reported to be similar to that in healthy controls, and therefore parotid saliva cannot provide diagnostically relevant information for this disease (Blomfield et al., 1976).

· Saliva from CF patients was found to contain an unusual form of epidermal growth factor (EGF). The EGF from these patients demonstrated poor biological activity compared with EGF from healthy controls. It was suggested that this EGF anomaly might contribute to the pathology of CF (Aubert et al., 1990).

· Further, abnormally elevated levels of prostglandins E2 (PGE2) were detected in the saliva of CF patients as compared with that of healthy controls (Rigas et al., 1989).

Coeliac disease is a congenital disorder of the small intestine that involves malabsorption of gluten. Gliadin is a major component of gluten.

· Serum IgA antigliadin antibodies (AGA) are increased in patients with coeliac disease and dermatitis herpetiformis.

· Measurement of salivary IgA-AGA has been reported to be a sensitive and specific method for the screening of coeliac disease, and for monitoring compliance with the required gluten-free diet (al-Bayaty et al., 1989; Hakeem et al., 1992).

21-Hydroxylase deficiency is an inherited disorder of steroidogenesis which leads to congenital adrenal hyperplasia. In non-classic 21-hydroxylase deficiency, a partial deficiency of the enzyme is present (Carlson et al., 1999).

· Early morning salivary levels of 17-hydroxyprogesterone (17-OHP) were reported to be an excellent screening test for the diagnosis of non-classic 21- hydroxylase deficiency, since the salivary levels accurately reflected serum levels of 17-OHP.

AUTOIMMUNE DISEASES—SJÖGREN'S SYNDROME

is an autoimmune exocrinopathy of unknown etiology.

The majority of patients are women

A reduction in lacrimal and salivary secretions is observed, associated with keratoconjunctivitis sicca and xerostomia. The presence of these two phenomena leads to a diagnosis of primary SS.

In secondary SS, a well-defined connective tissue disease (most commonly rheumatoid arthritis or systemic lupus erythematosus) is present in addition to the xerostomia and/or the keratoconjunctivitis (Schiødt and Thorn, 1989; Thorn et al., 1989).

In addition to involvement of the salivary and lacrimal glands, SS may also affect the skin, lungs, liver, kidneys, thyroid, and nervous system (Talal, 1992).

The diagnostic criteria for SS are still uncertain, and a single marker that is associated with all cases does not exist.

The accepted procedure for the diagnosis of the salivary involvement of SS is a biopsy of the minor salivary glands of the lip.

· SS is characterized by the presence of a lymphocytic infiltrate (predominantly CD4+ T-cells) in the salivary gland parenchyma (Daniels, 1984; Daniels and Fox, 1992).

· A low resting flow rate and abnormally low stimulated flow rate of whole saliva are also indicators of SS (Sreebny and Zhu, 1996a).

· Serum chemistry can demonstrate polyclonal hypergammaglobulinemia and elevated levels of rheumatoid factor, antinuclear antibody, anti-SS-A, and anti-SSB antibody (Atkinson et al., 1990; Fox and Kang, 1992). The immunologic mechanisms involved in the pathogenesis of the disease appear also to involve B-cells (the majority of lymphomas associated with SS are of the B-cell type), salivary epithelial cells, an activated mononuclear cell infiltrate, cytokines, and adhesion molecules (Fox and Speight, 1996).

· In sialochemistry – a consistent finding is increased concentrations of sodium and chloride. This increase is evident in both whole and gland specific saliva (Tishler et al., 1997).

· In addition, elevated levels of IgA, IgG, lactoferrin, and albumin, and a decreased concentration of phosphate were reported in saliva of patients with SS (Ben-Aryeh et al., 1981; Stuchell et al., 1984).

· Increased salivary concentrations of inflammatory mediators—i.e., eicosanoids, PGE2, thromboxane B2, and interleukin-6—have been reported (Tishler et al., 1996a,b).

· SS is characterized by autoantibodies to the La and Ro ribonucleoprotein antigens. These autoantibodies have been shown to target intracellular proteins which may be involved in the regulation of RNA polymerase function (Tan, 1989).

· Autoantibody, especially of the IgA class, can be synthesized in salivary glands and can be detected in the saliva of SS patients prior to detection in the serum (Horsfall et al., 1989).

· Saliva has also been reported to contain IgG autoantibody, while serum contained primarily IgG and IgM autoantibody (Ben-Chetrit et al., 1993).

Although variations in these cut-off values between clinicians may lead to differences in sensitivity and specificity in the diagnosis of SS, the quantitative evaluation of resting and stimulated saliva is a simple, non-invasive method of screening for patients who may have SS.

Reduced salivary flow, although not pathognomonic for SS, is of clinical importance and can lead to a variety of oral signs and symptoms, such as progressive dental caries, fungal infections, oral pain, and dysphagia (Daniels and Fox, 1992).

Dentists are normally the first to encounter these patients. Affected individuals should be referred for a comprehensive evaluation of the cause for the reduced salivary flow.

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 (Hainaut and Vahakangas, 1997; Tarapore and Fukasawa, 2000).

As a result, accumulation of inactive p53 protein is observed, which in turn may lead to the production of antibodies directed against this protein (Bourhis et al., 1996).

These antibodies can be detected in sera of patients with different types of malignancies (Lubin et al., 1995).

p53 antibody can also be detected in the saliva of patients diagnosed with oral squamous cell carcinoma (SCC), and can thus assist in the early detection of, and screening for, this tumor (Tavassoli et al., 1998).

Defensins are peptides which possess antimicrobial and cytotoxic properties.

They are found in the azurophil granules of polymorphonuclear leukocytes (PMNs; Lichtenstein et al., 1986; Lehrer et al., 1991).

Elevated levels of salivary defensin-1 were found to be indicative of the presence of oral SCC.

Elevated levels of recognized tumor markers c-erbB-2 (erb) and cancer antigen 15-3 (CA15-3) were found in the saliva of women diagnosed with breast carcinoma

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.

Tumor markers that can be identified in saliva may be potentially useful for screening for malignant diseases. Salivary diagnosis may be part of a comprehensive diagnostic panel that will provide improved sensitivity and specificity in the detection of malignant diseases and will assist in monitoring the efficacy of treatment. Additional studies are certainly required to determine which salivary markers can be used for these diagnostic purposes, and to determine their diagnostic value in comparison with other, more established, diagnostic tests.

INFECTIOUS DISEASES

DISEASES

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 saliva produced 97% sensitivity and 94% specificity in detection of the disease. The 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 (Li et al., 1996).

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 (Schultsz et al., 1992).

Pigeon breeder's disease (PBD) is an interstitial lung disease induced by exposure to antigens derived from pigeons. Measurement of salivary IgG against these antigens may assist in the evaluation of patients with this disease.

The detection of pneumococcal C polysaccharide in saliva by ELISA may offer a valuable complement to conventional diagnostic methods for pneumococcal pneumonia. Detection of this antigen in saliva demonstrated a sensitivity of 55% and specificity of 97%.

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, which in turn may serve as a screening mechanism for individuals at risk for Lyme disease (Schwartz et al., 1991).

Specific antibody to Taenia solium larvae in serum demonstrated greater sensitivity than antibody in saliva for identification of neurocysticercosis (100% vs. 70.4%, respectively). However, considering the simple and non-invasive nature of saliva sampling, it was suggested that saliva could be used in epidemiologic studies of this disease (Feldman et al., 1990).

SALIVARY ENZYMES

Diagnostic laboratory tests of serum are routinely used in evaluation of many systemic disorders. In contrast, diagnosis of periodontal disease relies primarily on clinical (GI, BOP,PD) and radiographic parameters (alveolar bone loss).

A response of an organism to the periodontal infection includes production of several enzyme families which are released from stromal, epithelial, inflammatory or bacterial cells.

The analysis of these enzymes in salivary secretion, as well as in the gingival crevicular fluid, can contribute to clarification of the pathogenesis and to improvement of making a prompt diagnosis of the periodontal disease.

Leading roles in this sense have the enzymes of tissue degradation - elastase, collagenase, gelatinase, proteinase.

Their activity can be proved in saliva, within some normal limits, as these enzymes are determined even in blood of healthy persons. However, if a periodontal tissue becomes sick, or its cells become damaged, due to edema or destruction of a cellular membrane, i.e. of a cell as a whole, these intracellular enzymes are increasingly being released into the gingival crevicular fluid and saliva where their activity can be measured. Due to this, these enzymes can be biochemical markers of the functional condition of periodontal tissues

Those particularly relevant in this group of enzymes are:

1. aspartate and alanine aminotransferases (AST and ALT),

2. lactate dehydrogenase (LDH),

3. gamma-glutamyl transferase (GGT),

4. creatine kinase (CK)

5. alkaline phosphatase (ALP),

6. acidic phosphatase (ACP).

LDH and AST can help monitor the progression of the periodontal disease. These enzymes appear to be useful to test the activity of periodontal disease or to measure the effectiveness of periodontal therapy

CK, LDH, AST, ALT and GGT are intracellular enzymes included in metabolic processes of cells and they are mostly present in cells of soft tissues - indicates the pathological changes located in soft tissues only, primarily in gingiva what could coincide with the initial stage of periodontal disease

ALP and ACP are intracellular enzymes present in most of tissues and organs, particularly in bones. Their increased activity in saliva is probably the consequence of destructive processes in the alveolar bone in advanced stages of development of periodontal disease

The activity of these enzymes in saliva can be of useful for the assessment of efficiency of changing the therapy in curing periodontal disease (2-4).

Previous studies mainly investigated the activities of these enzymes in gingival crevicular fluid, which is in a much closer contact with periodontal tissues and, due to this, it surely much better reflects the occurrences in them. However, the problem with the gingival crevicular fluid is in that the technique of collecting is rather complicated and hardly feasible in practice.

Contrary to the gingival crevicular fluid, there is plenty of saliva, the procedure of its sampling is much easier and more bearable for the patient and, the same enzymes as those in the gingival crevicular fluid can be detected. Becouse of the simple and non-invasive method of collection, salivary diagnostic tests apper to hold promise for the future

RESEARCH APPLICATIONS

Saliva already is used to aid in the diagnosis of dental disease. Examples include caries risk assessment, periodontal disease genotypes, and identification markers for periodontal disease, salivary gland disease and dysfunction, and candida infections.

Salivary collections are used for diagnostic determinants for viral diseases, sarcoidosis, tuberculosis, lymphoma, gastric ulcers and cancers, liver dysfunction, and Sjogren’s syndrome.

Saliva also is being used to monitor levels of polypeptides, steroids, antibodies, alcohol, and various other drugs.

Research currently is being conducted to

1. determine the value of saliva as a diagnostic aid for cancer and preterm labor.

2. regenerative properties and functions of growth factors found in saliva, such as epidermal growth factor and transforming growth factor. Evidence suggests that these growth factors play a role in wound healing and the maintenance of oral and systemic health.

The multifunctional roles of salivary components continue to represent a very focused area of dental research.

· Can the redundant and synergistic effects of salivary proteins be used to further enhance remineralization?

· Could the salivary antibacterial factors be targeted to positively alter the biofilm community in plaque?

· Can salivary constituents more selectively control bacterial adherence and aggregation?

· Can the buffering system of saliva effectively and selectively be enhanced?

· Can salivary components be reproduced or replaced by new developments in artificial saliva?

Questions such as these are being addressed through continuing research efforts.

CONCLUSIONS

Many areas of research involving salivary components and functions are in progress for local and systemic disease diagnosis, treatment, and prevention.

The value of saliva undoubtedly will continue to increase because it serves as an easily collected, noninvasive source of information.

Reflective of the status of health in the body, salivary samples can be analyzed for:

(1) tissue fluid levels of naturally, therapeutically, and recreationally introduced substances;

(2) emotional status;

(3) hormonal status;

(4) immunologic status;

(5) neurologic status;

(6) nutritional/metabolic influences

REFERENCES

1. Clinical Periodontology 10th Edition; Carranza,Newmann.

2. Shafers textbook of oral pathology. 5^th Edtm

3. Burkitt’s textboof of oral medicine. 11^th edtn

4. Periodontology 2000 volume 34: 2004

5. Tencate’s Oral histology 6th edition

6. J. Clinical Periodontology 2003;30:752-755

7. J. Clinical Periodontology 2000,27:453-465

8. J. Periodontal Research 1990,1983

9. J. Oral Pathology Medicine 1990.

10. Dentomaxillofac Radiol 2007;36:59-62. T Bar, A Zagury, D London, R Shacham, and O Nahlieli.

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