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Scope of saliva as a diagnostic fluid/prosthodontic courses


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Scope of saliva as a diagnostic fluid/prosthodontic courses

  1. 1. Scope of saliva as a diagnostic fluid INDIAN DENTAL ACADEMY Leader in continuing Dental Education
  2. 2. contents • Introduction • Composition of saliva • Collection of saliva • Saliva as diagnostic fluid • conclusion
  3. 3. • “Saliva is the clear mucoserous secretion formed mainly in salivary glands which has lubricative, cleansing & anti microbial, excretory and digestive functions”.
  4. 4. Based on their anatomical size  MAJOR SALIVARY GLANDS Parotid gland 30% Submandibular gland 60% Sublingual gland 5%  MINOR SALIVARY GLANDS 1% Buccal Labial Lingual Palatine Glossopalatine
  5. 5. BASED ON HISTO CHEMICAL NATURE OF SECRETION SEROUS, MUCOUS, MIXED  Parotid glands  Purely serous  SubmandPredominantlyserousMixed  Sublingual  Pre. mucousMixed  Labial,Buccal Pre.mucousMixed  Palatine, GlossopalatinePurely mucous  Anterior lingual glands  chiefly mucous  Posterior part of the tongue mixed  Von Ebner’s GlandsPurely serous
  7. 7. “ ORGANIC COMPONENTS 1)Salivary proteins : Mucins, statherins, histatins, immunoglobulin etc. 2) Digestive enzymes: α – amylase, lipase, peroxidase, lactoferrins, cystatins. 3) Carbonic anhydrase 4) Intrinsic growth factors: EGF , TGF α β 5) Circulating adrenal glucocorticoids: Coritsol. 6) Sex hormones in blood: Estriol. 7) Blood glucose in lower levels. 8) Blood group substances from A B O groups.
  8. 8. INORGANIC COMPONENTS Mainly •Sodium (Na +), •Potassium (K +) •Hydroxyappitite & calcium phosphate salts. •Chloride (Cl -) , Carbonic acid (Hco 3 - ) Smaller amounts •Magnesium (Mg 2+) •Thiocynate (SCN -) •Fluoride and Iodine
  9. 9. Saliva also contains constituents that do not originate in the salivary gland • including gingival crevicular fluid • serum transduate from the mucosa and sites of inflammation • epithelial and immune cells • microorganisms.
  10. 10. Factors affecting salivary composition
  11. 11. Normal salivary flow • In general, healthy adults produce 500–1500 mL of saliva per day, at a rate of approximately 0.5 mL/min • USFR < 0.1 mL/min • SFR < 0.7 Ml/min • are considered abnormally low, indicative of salivary gland hypofunction.
  12. 12. • When a person is in a resting state, saliva production is largely produced by the submandibular gland ( 2/3rd ) • In contrast, when saliva production is stimulated either via chewing gum or plastic (e.g. parafilm), or through acid stimulation, most of the saliva produced is from the parotid gland( ½)
  13. 13. Factors affecting salivary flow • Patient position • Hydration • Diurnal • Seasonal variation
  14. 14. TYPES OF SALIVA • Gland specific : The collection and evaluation of the secretions from the individual salivary glands • primarily useful for the detection of gland-specific pathology, i.e., infection and obstruction. • Whole saliva: mixture of oral fluids ,includes • secretions from both the major & minor salivary glands • several constituents of non-salivary origin such as (GCF) • expectorated bronchial and nasal secretions, • serum and blood derivatives from oral wounds, • bacteria & their products, viruses, fungi, • desquamated epithelial cells, cellular components, food
  16. 16. Precautions to be taken before sample collectionAvoid • alcohol for 12 hrs & dairy products for 20 min • foods with high sugar or acidity, or high caffeine content before sample collection. (may lower ph or increase bacterial growth) • Do not eat a major meal within 60 minutes before sample collection. • Document consumption of alcohol, caffeine, nicotine, & any medication
  17. 17. • Rinse mouth with water to remove food residue before sample collection, and swallow to increase hydration. • Wait at least 10 minutes after rinsing before collecting saliva to avoid sample dilution. • It is also advisable to document the physical activity level of research participants and the presence of oral diseases.
  18. 18. BLOOD CONTAMINATION OF SALIVA • Even an invisible amount of blood contamination has the potential to falsely elevate salivary analyte levels • participants should not brush their teeth within 45 minutes • Dental treatment should not be performed within 48 hours prior to sample collection. • Participants should be screened for oral health problems or injuries. • Saliva samples visibly contaminated with blood should be discarded and
  19. 19. • METHODS FOR COLLECTING WHOLE SALIVA • Draining • Suction • Spitting • Absorbent (sponge) method
  20. 20. SPITTING METHOD • In spitting method pt allows saliva to accumulate in mouth and then expectorates into a preweighed tube usually once every 60sec for 5 to 15 min ABSORBENT METHOD • In absorbent method a pre weiged gauge sponge is placed in pts mouth for a set amount of time.
  21. 21.
  22. 22. • If stimulated whole saliva collection is required • Standardised method of stimulation should be used • Chewing unflavoured gum base • Inert material like paraffin • Rubber band • At a controlled rate usually 60 tyms/min is reliable & reproducible means • 2% citric acid can also be placed on tongue at 30 sec interval.
  23. 23. Lashley cup/ Carlson – Crittenden Collector • Used for parotid • designed by carlson - crittenden • 2 metal cups with 2 outlet tubes • Inner cup collects saliva • Outer cup connected to vacuum • Basic design was modified by lashley
  24. 24. • Modified carlson-crittenden collector/ lashleys cannula • Single cup with two concentric chambers • The inner chamber can be positioned over parotid duct orifice for collection of saliva • While suction applied to outer chamber holds cup in place.
  25. 25. • Parotid saliva can be stimulated by applying 1ml of 2% citric acid to dorsum of tongue every 30 sec • Depending on age & health status of subject flow rate is usually 1 to 3 ml/min
  26. 26. Schneyers apparatus
  27. 27. Segregator
  28. 28. SCOPE OF SALIVA AS DIAGNOSTIC FLUID Though saliva lacks Drama of blood Sincerity of sweat Emotional component of tears It is a unique fluid Which harbors a wide spectrum of Proteins, nucleic acids, electrolytes, and hormones that originate from multiple local and systemic sources. saliva reflects the body’s health and well-being
  29. 29. • Salivary diagnosis is an increasingly important field in • Dentistry • Physiology • Internal Medicine, • Endocrinology • Pediatrics • Immunology • Clinical Pathology, • Forensic Medicine, • Psychology • Sports Medicine
  30. 30. ADVANTAGES OF SALIVA ( WHEN COMPARED TO SERUM ) AS DIAGNOSTIC FLUID • collected noninvasively by individuals with modest training. • Easy to store & transport • Cost effective approach for the screening of large populations. • Multiple collections can be performed without imposing too much discomfort on the donor
  31. 31. • Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. • Minimal risk of contracting infections during saliva collection • saliva can be used in clinically challenging situations, such as obtaining samples from children , handicapped or anxious patients in whom blood sampling could be a difficult act to perform.
  32. 32. DISADVANTAGES OF SALIVA AS DIAGNOSTIC TOOL • Diurnal /circadian variations of certain biomolecules present in saliva • It does not always reliably reflect the concentrations of these molecules in serum. Salivary composition can also be influenced by • method of collection • Degree of stimulation of salivary flow
  33. 33. • Saliva contains analytes in concentrations that are 1000-fold less than those in blood. • Sensitive detection systems are needed.
  34. 34. • Most of the biomolecules that are present in saliva are either synthesized in situ in the salivary glands and/or transported from blood capillaries into saliva • understanding of the biomolecules present in saliva during a normal healthy physiological state, as opposed to a pathological condition, required in order for saliva to become a sample of choice for diagnostic and treatment purposes.
  35. 35. Transfer of biomolecules from blood to saliva DIFFUSION ACTIVE TRANSPORT ULTRA FILTRATION TRANSUDATION
  36. 36. DIFFUSION • common route for substances to migrate from blood to saliva is via unaided or passive diffusion • capillaries surrounding the glands are quite porous for many small molecules. • A serum molecule must cross 5 barriers to reach saliva : 1. The Capillary Wall 2. The Interstitial Space; 3. The Basal Cell Membrane Of The Acinus Cell / Duct Cell; 4. The Cytoplasm Of The Acinus Or Duct Cell 5. The Luminal Cell
  37. 37. • Ability of molecule to pass depends on its size, and electrical charge • If a molecule is polar in nature, & separates into charged ions while in solution, it is difficult to pass • steroid hormones are relatively small in size, & composed of fatty acids, so they tend to pass relatively easily by diffusion. • Molecules that are bound to large carrier proteins, such as serum albumin, are too big to enter by this route
  38. 38. ACTIVE TRANSPORT • route used by secretory IgA (SIgA). • polymeric IgA, which is secreted by B-lymphocyte cells in close proximity to salivary cells, is then bound by IgA receptors present on acinus cells, & then gets released into saliva
  39. 39. Lumen
  40. 40. Ultrafiltration • Filtration through the spaces between acinus and ductal cells. • Also through gap junctions between cells of secretory units ( intracellular nexus) • To follow this type of transportation into saliva, the molecules must be relatively small.( < 1900da) • such as water, ions, catecholamines, and steroids • Sulfated steroids, estroil steroids cannot pass through pl bilayer due to their charge
  41. 41. Transudation: • Transudation of plasma components into oral cavity either from crevicular fluid/ directly from mucosa • Plasma albumin in saliva mostly occurs through this.
  42. 42. • Diagnosis of oral diseases • Caries activity tests • Systemic diseases Hereditary Autoimmune Malignancy Infectious Cvs, renal • Drug monitoring • Monitoring level of hormones • Forensic odontology
  43. 43. Diagnosis of Oral Disease • Quantitative alterations in saliva may be a result of medications. At least 400 drugs may induce xerostomia. • Diuretics, antihypertensives, antipsychotics, antihistamines, antidepressants, anticholinergics, antineoplastics, and recreational drugs such as opiates, amphetamines, barbiturates, hallucinogens, cannabis, and alcohol have been associated with a reduction in salivary flow
  44. 44. • Reduced salivary flow may lead to oral problems like progressive dental caries, fungal infection, oral pain, and dysphagia. • Saliva can be used for the detection of oral candidiasis, and salivary fungal counts may reflect mucosal colonization.
  45. 45. Dental caries • Saliva may also be used for the monitoring of oral bacteria. • Anaerobic bacteria can survive in saliva, and can utilize it as a growth medium • Studies have shown that salivary parameters such as flow rate, viscosity, ph, buffering capacity were decreased in subjects with high risk of caries • So saliva testing is an important part in routine protocol while diagnosing & treating dc.
  46. 46. Caries activity tests Lactobacillus colony count test • Introduced by HADLEY in 1933 • Estimates no of acidogenic bacteria • Saliva is collected by chewing paraffin wax for 3min ( before breakfast) • specimen is vigourously Shaken for 2 minutes. • Diluted with Distilled Water • Diluted sample are spread evenly on Petri dishes • Containing 20ml of cooled Liquefied Agar ( Rogasa’s SL agar plate )
  47. 47. • The plates are incubated for 3to4 days at 37C • Number of Lactobacillus colonies are counted No of organisms per cc Symbolic designation Degree of caries activity suggested 0 – 1000 1000- 5000 +/- + Little or None Slight 5000- 10,000 ++ Moderate More than 10,000 +++or++++ Marked
  48. 48. CALORIMETRIC SNYDER TEST • The ability of salivary microorganism to form organic acids from a carbohydrate medium • The glucose agar medium contains an indicator dye, Bromocresol green. • Changes color from green to yellow in the range of pH 5.4 to 3.8 • Salivary sample is collected & 0.2 cc of saliva is pipetted into the melted medium at 50c • The incubation period is up to 72 h at 37c • The rate of color change from green to yellow is indicative of the degree of caries activity.
  49. 49. 24 hours If yellow 48 hours If yellow 72 hours If yellow Marked caries susceptibility Definite caries susceptibility Limited caries susceptibility If Green Continue to incubate & observe at 48 h If Green Continue to incubate & observe at 72 h If Green Caries inactive
  50. 50. Streptococcus mutans level in Saliva • Measures the no of streptococcus mutans CFU per unit volume of saliva. Procedure • Sample of organisms is obtained by the use of tongue blades ( Wooden spatulas) • Then pressed against Streptococcus mutans selective MSB ( Mitus salivarius Bacitracin ) Agar in special Petri dishes.
  51. 51. Interpretation • Level of Streptococcus Mutans>10 /ml of saliva = Unacceptable. Disadvantages • Difficulty of Distinguishing b/w a carrier state and cariogenic state • SM may constitute less than 1% of total flora of plaque • SM tends to be located at specific site only.
  52. 52. ENAMEL SOLUBILITY TEST • When Glucose is added to the saliva containing powdered Enamel, Organic acids are formed. • this acids Decalcify the Enamel • Resulting in an increase in the amount of soluble calcium in the saliva – glucose – enamel mixture. • Extent of increase in calcium is a direct measure of the degree of caries susceptibility.
  53. 53. SALIVARY REDUCTASE TEST • Measure the activity of the reductase enzyme present in salivary bacteria. • Kit – Treatex. • Saliva is collected in a plastic container. • Sample is then mixed with the dye Diazoresocinol • Color changes and the caries conduciveness • Reading is taken after 15 minutes • No incubation procedure
  54. 54.
  55. 55. ALBAN TEST Materials • Snyder test agar • A small scale to measure 60 grams • A 2 liter Pyrex glass to melt the medium • A funnel to dispense the medium into test tubes • 100mm,16mm test tubes with screw caps.
  56. 56. Procedure • 60 grams of Snyder test agar is placed in 1 liter of water • The suspension is brought to a boil over a low flame. • Melted agar is distributed using about 5ml/tube • Autoclaved for 15 min • Allowed to cool • Stored in a refrigerator
  57. 57. • 2 tubes of alban medium are taken from the refrigerator. • Pt is asked to expectorate a small amount of saliva directly into the tubes • Labeled • Incubated at 98.6F for up to 4 days • The tubes are observed daily
  58. 58. Scale for Scoring • No color change • Beginning Color change = ‘ + ’ ( from top of medium down ) • One half color change = ++ • ¾ color change = +++ • Total color change = ++++ From bluish green to definite yellow pH 5 to 4
  59. 59. • Reading -ve - Labelled Negative • All other readings - +ve • Slower change is - Improved • Faster change - Worse • Consecutive readings are nearly identical – No change
  60. 60. Periodontitis • Specific immunoglobulins in saliva directed toward periodontal pathogens have been identified. • saliva from treated periodontitis patients had higher IgA and IgG levels to periodontalpathogens porphyromonas. gingivalis and Treponema. denticola than as compared to saliva from control subjects • Patients with periodontal disease have demonstrated high levels of this peroxidase enzyme in saliva
  61. 61. • Elevated Ca concentration in saliva • Volatile sulfur compounds, primarily hydrogen sulfide and methylmercaptan, are associated with oral malodor. • Salivary volatiles have been suggested as possible diagnostic markers and contributory factors in periodontal disease. • . Circulating C-reactive protein may reach saliva via GCF or the salivary glands. High levels of C-reactive protein have been associated with chronic and aggressive periodontal diseases
  62. 62. Recurrent aphthous stomatitis : • Evidence shows that there is increased oxidative stress • levels of both plasma and salivary antioxidants are decreased in patients with RAU.
  63. 63. Systemic diseases Hereditary Autoimmune Malignancy infectious
  64. 64. Cystic fibrosis (CF) : • lethal, congenital, genetic disease of unknown etiology. • Defective regulation of ion and water transport in exocrine glands and possibly also in other epithelial cells has a central role in the pathogenesis of this disease. • Abnormally high calcium levels in some secreted fluids and some cells of CF patients.
  65. 65. • Organs most commonly affected are • Sweat glands – produce inc conc of nacl • Pancreas - pancreatic insufficiency • Lungs – develops copd • Saliva of cf pts contain inc ca levels • A factor produced by CF fibroblasts in culture can increase the calcium concentration in healthy cells, although this may be an indirect effect
  66. 66. • Inc in calcium protein aggregates in sub mand saliva is seen causing turbidity • Saliva contains an un usual epidermal growth factor • Inc salivary pge2 is detected • Parotid saliva donot demonstrate characteristic changes
  67. 67. Sjogrens syndrome • There was a reduced flow rate ( <0.1 ml/min) • whole saliva flow rate (Sreebny and Zhu, 1996) and gland-specific sialometry and sialochemistry (Kalk et al., 2002) could be used to provisionally diagnose SS • Measuring submandibular/sublingual flow rates may contribute to an early diagnosis of ss.
  68. 68. Sialochemistry reveals • The Na+ conc in the parotid glandular saliva is six fold higher in SS patients as compared to non-SS • Reduced phosphate level compared to normal controls. • Increase in IgA, IgG and lactoferrin. • Albumin concentration was also elevated • Inc beta 2 microglobulin • Inc conc of cystatin c & s
  69. 69. Renal disease • In patients with CRF increase in saliva urea, creatinine is found. • colormetric test strips were used to monitor salivary nitrate and uric acid before and after hemodialysis. • It was suggested that a salivary test could be used by patients to decide when dialysis is required, thereby eliminating unnecessary visits to a dialysis clinic
  70. 70. • Salivary phosphate has been successfully used as a clinical biomarker forhyperphosphatemia, which is an important contributor to cardiovascular calcification in chronic renal failure (CRF) • Evaluation of phosphate levels in saliva correlated positively with serum creatinine & gfr . Thus, salivary phosphate may provide a better marker than serum phosphate for the initiation of treatment of hyperphosphatemia in CRF and HD.
  71. 71. cvs • C-reactive protein (CRP) can be monitored in salivary samples, • but CRP remains a non-specific inflammatory response factor that increases in many conditions including periodontal disease • markers include CRP, myoglobin & myeloperoxidase, in combination with an ECG, showed a highly significant correlation with myocardial infarct patients as compared to healthy controls. • Robust clinical studies are required to validate salivary biomarkers for CVD
  72. 72. Infectious diseases Bacterial viral fungal protozoal spirochetal
  73. 73. • Helicobacter pylori infection is associated with peptic ulcer disease and chronic gastritis. • Infection with this bacterium stimulates the production of specific IgG antibody. • An ELISA test for the detection of IgG antibody in serum produced 97% sensitivity and 94% specificity in detection of the disease • sensitivity of saliva samples tested for the presence of H. pylori DNA by (PCR) assay is 84%
  74. 74. • 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
  75. 75. • 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
  76. 76. • Lyme disease is caused by the spirochete Borrelia burgdorferi and is transmitted to humans by blood- feeding ticks. • The detection of anti-tick antibody in saliva has potential as a biologic marker of exposure to tick bites, • This serve as a screening mechanism for individuals at risk for Lyme disease
  77. 77. • Antibodies against viruses and viral components can be detected in saliva and can aid in the diagnosis of acute viral infections, congenital infections, and reactivation of infection • Saliva was found to be a useful alternative to serum for the diagnosis of viral hepatitis. Acute hepatitis A (HAV) and hepatitis B (HBV) were diagnosed based on the presence of IgM antibodies in saliva
  78. 78. • Analysis of oral fluid samples collected with Orasure® provided an excellent method for the diagnosis of viral hepatitis B and C. • Sensitivity and specificity of 100% for the detection of antibodies for both diseases in oral fluid in comparison with serum antibodies were reported
  79. 79. • Saliva may also be used for determining immunization ,detecting infection with measles, mumps, and rubella • detection of antibodies in oral fluid samples produced sensitivity and specificity of 97% and 100% for measles, • 94% and 94% for mumps • 98% and 98% for rubella, respectively, • In comparison with detection of serum antibodies for these viruses
  80. 80. • The shedding of herpesviruses (human herpesvirus –8, cytomegalovirus, and Epstein-Barr virus) in nasal secretions and saliva of infected patients has been reported • Other investigators suggested that reactivation of herpes simplex virus type-1 (HSV-1) is involved in the pathogenesis of Bell's palsy and reported that PCR-based identification of virus in saliva is a useful method for the early detection of HSV-1 reactivation in patients with Bell's palsy. • The shed HSV-1 virus was detected in 50% of patients with Bell's palsy in comparison with 19% in healthy controls
  81. 81. • Dengue is a mosquito-transmitted viral disease. • Primary infection of the virus may lead to a self-limiting febrile disease • secondary infection may cause serious complications like dengue hemorrhagic fever or dengue shock syndrome. • Salivary levels of anti-dengue IgM and IgG demonstrated sensitivity of 92% and specificity of 100% in the diagnosis of primary and secondary infection, and salivary levels of • IgG proved useful in differentiating between primary and secondary
  82. 82. • Studies have demonstrated that the diagnosis of infection with the human immunodeficiency virus (HIV) based on specific antibody in saliva is equivalent to serum in accuracy, and therefore applicable for both clinical use and epidemiological surveillance • Antibody to HIV in whole saliva of infected individuals, which was detected by ELISA and Western blot assay, correlated with serum antibody levels • As compared with serum, the sensitivity and specificity of antibody to HIV in saliva for detection of infection are between 95% and 100%
  83. 83. • Several salivary and oral fluid tests have been developed for HIV diagnosis. • Orasure® is a testing system that is commercially • available in the United States and can be used for the diagnosis of HIV. • The test relies on the collection of an oral mucosal transudate (and therefore IgG antibody). • IgG antibody to the virus is the predominant type of anti-HIV immunoglobulin
  84. 84. Malignancy • Salivary analysis may aid in the early detection of certain malignant tumors. • p53 is a tumor suppressor protein which is produced in cells exposed to various types of DNA-damaging stress. • Inactivation of this suppressor through mutations and gene deletion is considered a frequent occurrence in the development of human cancer • Abs to this inactive p53 are found in sera & saliva of pts with tumors • P53 abs seen in saliva of
  85. 85. • Defensins are peptides which possess antimicrobial and cytotoxic properties. • They are found in the azurophil granules of pmn • Elevated levels of salivary defensin-1 were found to be indicative of the presence of oral SCC. • Higher concentrations of salivary defensin-1 were detected in patients with oral SCC • A high-positive correlation was observed between salivary defensin-1 levels and serum levels of SCC-related antigen
  86. 86. • salivary nitrate, nitrite, and nitrosamine may be related to the development of oral and gastric cancer • Increased consumption of dietary nitrate and nitrite is associated with elevated levels of salivary nitrite. • Higher levels of salivary nitrate and nitrite, and increased activity of nitrate reductase, were found in oral cancer patients compared with healthy individuals, and were associated with an increased odds ratio for the risk of oral cancer
  87. 87. • elevated levels of tumor markers c-erbB-2 (erb) and cancer antigen 15-3 (CA15-3 were found in the saliva of women diagnosed with breast carcinoma, as compared with patients with benign lesions and healthy controls • CA 125 is a tumor marker for epithelial ovarian cancer. Elevated salivary levels of CA 125 were detected in patients with epithelial ovarian cancer as compared with patients with benign pelvic masses and healthy controls. A positive correlation was found between salivary and serum levels of CA 125.
  88. 88. Hormone levels • The majority of hormones enter saliva by passive diffusion across the acinar cells. • Salivary hormone levels represent the non-protein- bound (free) serum hormone levels. • Small molecules enter by ultra filtration • Some steroid hormones can be metabolized in the salivary epithelial cells by intracellular enzymes during transcellular diffusion, which can affect the availability of these hormones in
  89. 89. • Due to their lipid solubility, steroid hormones can be detected in saliva. • Salivary cortisol levels demonstrate excellent correlation with free serum cortisol levels • actual salivary cortisol levels are lower than the serum-free cortisol levels, possibly due to enzymatic degradation in the salivary epithelial cells during transcellular diffusion • Salivary cortisol levels were found to be useful in identifying patients with Cushing's syndrome & Addison's disease • monitoring the hormone response to physical exercise and the effect of acceleration
  90. 90. • Salivary aldosterone levels demonstrated a high correlation with serum aldosterone levels and increased aldosterone levels were found in both the serum and saliva of patients with primary aldosteronism (Conn's syndrome) • The sodium (Na) and potassium (K) conc of saliva are markedly affected by corticosteroids, especially aldosterone. • The Na/K ratio of stimulated whole saliva can be used in diagnosing and monitoring Cushing’s syndrome and Addison’s disease, primary aldosteronism
  91. 91. Testosterone and dehydroepiandrosterone have also been identified in saliva. Salivary concentrations were found to 1.5-7.5% of the serum concentrations of these hormones
  92. 92. • Estradiol can be detected in saliva in concentrations that are only 1-2% of serum concentrations. • These concentrations are similar to the serum concentrations of free estradiol, which can diffuse into saliva • Salivary estriol levels showed a very high correlation with serum levels of free estriol in pregnant women, and salivary estriol levels were suggested as a means for the assessment of feto-placental function • Decreased levels suggested a marker of fetal growth retardation.
  93. 93. • Salivary progesterone levels showed good correlation (r = 0.47-0.58) with serum levels during the menstrual cycle and reflected the free serum progesterone levels • Salivary progesterone levels can be useful for the prediction of ovulation
  94. 94. • Insulin can be detected in saliva, and salivary insulin levels have been evaluated as a means of monitoring serum insulin levels. • A positive correlation between saliva and serum insulin levels following a glucose tolerance test was reported for healthy subjects, niddm patients, and obese non-diabetic patients • Salivary insulin levels reached maximal values approximately 30 minutes after the serum levels
  95. 95. limitations of salivary analysis for hormone evaluation. • mostly lipid-soluble and hormones with small molecular weight can be detected in saliva. • For hormones that demonstrate a constant but low salivary-to- serum ratio, a sufficiently large sample volume or a more sensitive analysis method is required. • In addition, many hormones exhibit marked circadian variations. Therefore, timing of saliva collection may affect the results. • The salivary flow rate can also affect the concentrations of certain hormones. An increase in salivary flow rate will usually result in reduced concentrations of molecules that reach saliva by diffusion
  96. 96. • Changes in salivary flow rate may lead to changes in salivary pH. This may affect the entry into saliva of molecules according to their pka • Hormones in saliva can be degraded, among other ways, by enzymes native to saliva, enzymes derived from oral micro- organisms, and enzymes derived from leukocytes that enter the oral cavity from gingival sulcus. • These factors have to be considered when saliva is evaluated as an alternative for the evaluation of serum hormone levels.
  97. 97. Drugs • Passive diffusion across a concentration gradient is thought to be the major mechanism to account for the appearance of a drug in saliva. • Therefore, drugs which are not ionizable, or are not ionized within the pH range of saliva, are the most suited to salivary drug monitoring. • Due to their size, serum binding proteins do not cross the membrane. Therefore, only the unbound fraction of the drug (pharma active) in serum is available for diffusion into saliva
  98. 98. • Saliva is also useful for the monitoring of anti-epileptic drugs. • Salivary carbamazepine levels were found to be 38% of serum carbamazepine levels, and a positive correlation (r = 0.89) between salivary and serum carbamazepine levels was observed. • salivary levels of phenobarbital and phenytoin demonstrated excellent correlations with serum levels of these medications
  99. 99. • Cyclosporine is a neutral lipophilic molecule that enters saliva mostly by passive diffusion, and salivary levels of this drug reflect the serum levels of free cyclosporine. • Therefore, salivary cyclosporine levels may correlate better with serum levels of free, rather than total, cyclosporine • Similarly, salivary theophylline concentration demonstrated a better correlation with serum concentration of free theophylline (r = 0.85) than with serum concentration total theophylline
  100. 100. • Therapeutic drugs: • Antipyrine Metoprolol • Oxprenolol • Paracetamol • Phenytoin • Primidone • Procainamide • Quinine • Sulfanilamide • Theophylline • Tolbutamide •Caffeine •Carbamazepine •Cisplatin •Cyclosporine •Diazepam •Digoxin •Ethosuximide •Irinotecan •Lithium •Methadone
  101. 101. • Saliva can be used for evaluation of illicit drug use • Following drug use the appearance of the drug in saliva follows a time course that is similar to that of serum. In contrast, drugs appear at a later time point in urine. • Presence of drug and not their conc is found • Exception is ethanol. • Ethanol is not ionized in serum, is not proteinbound, and, due to its low molecular weight and lipid solubility, diffuses rapidly into saliva.
  102. 102. • Consequently, the saliva to- serum ratio is generally about 1 • Salivary ethanol concentration may be used as an index of the blood ethanol concentration, provided that the salivary sample is obtained at least 20 min following ingestion. • This will allow for absorption and distribution of alcohol, and prevent a falsely elevated reading due to the oral route of consumption
  103. 103. • Other recreational drugs that can be identified in saliva are • amphetamines, barbiturates, • benzodiazepines, cocaine, • Phencyclidine (PCP), and opioids • Saliva can also be used to detect recent marijuana use by means of radiommunoassay • D9-Tetrahydrocannabinol (D9-THC), a major psychoactive component of marijuana, can be detected in saliva for at least 4 hours after marijuana is
  104. 104. • Furthermore, saliva can be used to monitor tobacco smoking and exposure to tobacco smoke. • The major nicotine metabolite cotinine was investigated as an indicator of exposure to tobacco smoking. • Cotinine is tobacco-specific and has a relatively long half-life compared with nicotine • Salivary cotinine levels were found to be indicative of active and passive smoking • Salivary thiocyanate was also found to be an indicator of cigarette smoking
  105. 105. Saliva in forensics • Salivary test have been used for a wide variety of forensic studies. • Samples can be obtained from drinking glasses, cigarette butts, envelopes, and other sources and then used to detect blood-group substances or salivary genetic proteins (primarily proline-rich protein polymorphisms). • Approximately 85% of individuals secrete blood-group antigens in their saliva including A, B, H, and Lewis antigens that have been used for identification of individuals in both criminal cases and paternity law suits
  106. 106. • Saliva is often present at crime scenes, along with other body fluids, and since DNA is relatively stable in the dry state, these samples can be used to place an individual at the scene of a crime.
  107. 107. Conclusion • Saliva offers an alternative to serum as a biologic fluid that can be analyzed for diagnostic purposes. • Saliva is particularly useful for qualitative (detection of the presence or absence of a marker) rather than quantitative diagnosis, which makes it an important means for the detection of viral infection (especially HIV due to the non-invasive method of collection), past exposure and immunity, and the detection of illicit drug use
  108. 108. • Early studies using saliva as a diagnostic fluid were hampered by • lack of understanding on mech by which markers enter oral cavity • the difficulty in detection due to low levels • lack of attention to the method of collection • storage of samples prior to analysis These challenges have met as a result of • careful studies of salivary gland physiology, • development of sensitive amplification methods (e.g. ELISA, qRT-PCR), and
  109. 109. • Salivary proteome: The proteome is the protein complement of the genome • proteomics is analysis of the portion of the genome that is expressed. • The proteomes in body fluids are valuable due to their high clinical potential as sources of disease markers. • Collectively, 1166 salivary proteins have been identified: 914 from the parotid fluid and 917 from the combined submandibular and sublingual fluids
  110. 110. • Salivary transcriptome: Salivary transcriptome is an emerging concept. • They included mRNA molecules that cells use to convey the instructions carried by DNA for subsequent protein production. • This discovery presented a second diagnostic alphabet in saliva and opened a door to another avenue of salivary transcriptomic diagnostics • Other research groups, particularly from forensic sciences, are focusing on multiplex mRNA profiling for the identification of body fluids, including saliva
  111. 111. • potential advantages of salivary analysis for the diagnosis of systemic disease suggest that further studies are warranted.
  112. 112. References : • Tina Pfaffe,1 Justin Cooper-white,2,3 Peter Beyerlein. Diagnostic Potential Of Saliva : Current State And Future Applications. Clinical Chemistry 57:5 675–687 (2011) • Eliaz Kaufman And Ira B. Lamster. The Diagnostic Applications Of Saliva-- A Review. Crit Rev Oral Biol Med. 13(2):197-212 (2002)