1. Saliva

2. Composition of Saliva 97% to 99.5% water 97% to 99.5% water Minerals Minerals Electrolytes Electrolytes Buffers Buffers Enzymes (amylase, lysozyme, sialoperoxidase, lingual lipase, ribonuclease, deoxyribonuclease, kallikreins, and carbonic anhydrases) Enzymes (amylase, lysozyme, sialoperoxidase, lingual lipase, ribonuclease, deoxyribonuclease, kallikreins, and carbonic anhydrases) Enzyme inhibitors Enzyme inhibitors Growth factors Growth factors

3. Composition of Saliva Cytokines Cytokines R proteins which binds cobalamines (B 12 ) and keep them in absorbable form R proteins which binds cobalamines (B 12 ) and keep them in absorbable form Immunoglobulines (secretory immunoglobulin A, sIgA) Immunoglobulines (secretory immunoglobulin A, sIgA) Mucin Mucin Antimicrobial components (Histatins, Statherins, Proline- rich proteins, Lactoferrin) Antimicrobial components (Histatins, Statherins, Proline- rich proteins, Lactoferrin) Antibodies Antibodies

4. Functions SalivaryFamilies Anti-Bacterial Buffering Digestion Mineral-ization Lubricat- ion &Visco- elasticity TissueCoating Anti-Fungal Anti-Viral

5. Salivary Proteins Salivary proteins occurring in families, consisting of structurally closely related family members Salivary proteins occurring in families, consisting of structurally closely related family members Salivary protein biosynthesis starts with the transcription and translation of salivary protein genes in the glands Salivary protein biosynthesis starts with the transcription and translation of salivary protein genes in the glands Post-translational processing involving protein glycosylation, phosphorylation and proteolysis Post-translational processing involving protein glycosylation, phosphorylation and proteolysis Once glandular secretions enter the non-sterile oral environment, proteins are subjected to additional and continuous protein modifications, leading to extensive proteolytic cleavage, partial deglycosylation, and protein- protein complex formation Once glandular secretions enter the non-sterile oral environment, proteins are subjected to additional and continuous protein modifications, leading to extensive proteolytic cleavage, partial deglycosylation, and protein- protein complex formation

6. Mucins (mucus glycoproteins) Mucins are extra-cellular large highly glycosylated molecules having mucin domains Mucins are extra-cellular large highly glycosylated molecules having mucin domains Mucin domains are rich in the amino acids threonine, serine, and proline Mucin domains are rich in the amino acids threonine, serine, and proline Oligosaccharides are linked to the hydroxyl amino acids Oligosaccharides are linked to the hydroxyl amino acids Mucins are of two types; secreted and membrane bound Mucins are of two types; secreted and membrane bound Secreted mucins are gel-forming due to their polymeric nature Secreted mucins are gel-forming due to their polymeric nature

8. Mucins The major mucin of the gastrointestinal tract is called MUC2 and is produced by the intestinal goblet cells The major mucin of the gastrointestinal tract is called MUC2 and is produced by the intestinal goblet cells MUC2 can form insoluble mucus gels MUC2 can form insoluble mucus gels MUC2 has two central mucin domains and cysteine-rich domains at both the N- and C- terminal ends MUC2 has two central mucin domains and cysteine-rich domains at both the N- and C- terminal ends Addition of O-glycans starts in Golgi apparatus Addition of O-glycans starts in Golgi apparatus MUC2 forming polymers in late Golgi apparatus MUC2 forming polymers in late Golgi apparatus

9. Mucin Functions Tissue Coating Tissue Coating – Protective coating about hard and soft tissues – Concentrates anti-microbial molecules at mucosal interface Lubrication Lubrication – Align themselves with direction of flow (characteristic of asymmetric molecules) – Increases lubricating qualities (film strength)

10. Statherins: Statherins: – Inhibit mineralization and so help to prevent precipitation of calcium and phosphate on intact tooth surface. – Prevent crystallization of supersaturated calcium phosphate in the salivary duct and oral fluid Proline-rich Proteins (PRPs): Proline-rich Proteins (PRPs): – Coat the teeth with a thin layer (pellicle) that serves as a protective diffusion barrier on the tooth surface – Inhibit mineralization and so help to prevent precipitation of calcium and phosphate on intact tooth surface.

11. Anti-microbial activities of saliva Lactoferrin: Lactoferrin: – Nutritional immunity (iron starvation) – Some microorganisms (e.g., E. coli) have adapted to this mechanism by producing enterochelins. bind iron more effectively than lactoferrin bind iron more effectively than lactoferrin iron-rich enterochelins are then reabsorbed by bacteria iron-rich enterochelins are then reabsorbed by bacteria – Lactoferrin, with or without iron, can be degraded by some bacterial proteases. – In unbound state, a direct bactericidal effect

12. Anti-microbial activities of saliva Lysozymes: Lysozymes: – Present in numerous organs and most body fluids – Oral LZ is derived from at least four sources Salivary glands, phagocytic cells and gingival crevicular fluid (GCF) Salivary glands, phagocytic cells and gingival crevicular fluid (GCF) – Biological function Classic concept of anti-microbial activity of LZ is based on its muramidase activity (hydrolysis of beta (1-4) bond between N- acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer. Classic concept of anti-microbial activity of LZ is based on its muramidase activity (hydrolysis of beta (1-4) bond between N- acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer. Gram negative bacteria generally more resistant than gram positive Gram negative bacteria generally more resistant than gram positive

13. Anti-microbial activities of saliva Histatins: Histatins: – A group of small histidine-rich proteins – Potent inhibitors of Candida albicans growth Cystatins: Cystatins: – Are inhibitors of cysteine-proteases

14. Anti-microbial activities of saliva Salivary peroxidase systems: Salivary peroxidase systems: – Sialoperoxidase (SP, salivary peroxidase) Produced in acinar cells of parotid glands Produced in acinar cells of parotid glands Also present in submandibular saliva Also present in submandibular saliva Readily adsorbed to various surfaces of mouth, Readily adsorbed to various surfaces of mouth, enamel, salivary sediment, bacteria, dental plaque – Myeloperoxidase (MP) From leukocytes entering via gingival crevice From leukocytes entering via gingival crevice 15-20% of total peroxidase in whole saliva 15-20% of total peroxidase in whole saliva

15. Components of the peroxidase anti-microbial system Peroxidase enzymes (SP or MP) Peroxidase enzymes (SP or MP) Hydrogen peroxide (H 2 O 2 ) Hydrogen peroxide (H 2 O 2 ) – oral bacteria (facultative aerobes/catalase negative) produce large amounts of peroxide Thiocyanate ion (SCN - ) which is converted to hypothiocyanite ion (OSCN - ) by peroxidase Thiocyanate ion (SCN - ) which is converted to hypothiocyanite ion (OSCN - ) by peroxidase – salivary concentration is related to diet and smoking habits

16. Thiocyanate reactions Hydrogen peroxide (bacterial activity) + thiocyanate (saliva) → → → → oxidation product (hypothiocyanate) toxic to bacteria oxidation product (hypothiocyanate) toxic to bacteria

17. HOSCN/OSCN - -mediated cell damage Can oxidize sulfhydryl groups of enzymes Can oxidize sulfhydryl groups of enzymes Block glucose uptake Block glucose uptake Inhibit amino acid transport Inhibit amino acid transport Damage inner membrane, leading to leakage of cells Damage inner membrane, leading to leakage of cells Disrupt electrochemical gradients Disrupt electrochemical gradients

18. Amylases Two types; pancreatic (P-type) and salivary (S-type). Two types; pancreatic (P-type) and salivary (S-type). Alpha-amylases are calcium metalloenzymes Alpha-amylases are calcium metalloenzymescalciummetalloenzymescalciummetalloenzymes Acts on alpha-1,4- glycosidic bonds Acts on alpha-1,4- glycosidic bondsglycosidic bondsglycosidic bonds Full activity is displayed only in the presence of various anions, such as chloride, bromide, nitrate or cholate. Full activity is displayed only in the presence of various anions, such as chloride, bromide, nitrate or cholate. It is the only plasma enzyme normally found in urine It is the only plasma enzyme normally found in urine

19. Amylases There are 12 distinct phenotypes for the salivary isoenzyme and 6 for the pancreatic isoenzyme. There are 12 distinct phenotypes for the salivary isoenzyme and 6 for the pancreatic isoenzyme. Isoenzymes are products of two closely linked loci on chromosome 1 Isoenzymes are products of two closely linked loci on chromosome 1 Salivary amylase (ptyalin), is found in saliva and breaks starch down into maltose and dextrin Salivary amylase (ptyalin), is found in saliva and breaks starch down into maltose and dextrinmaltosedextrinmaltosedextrin Salivary amylase is inactivated in the stomach by gastric acid Salivary amylase is inactivated in the stomach by gastric acidstomachgastric acidstomachgastric acid Pancreatic α-amylase randomly cleaves the α (1-4) glycosidic linkages of amylose to yield dextrin, maltose or maltotriose. Pancreatic α-amylase randomly cleaves the α (1-4) glycosidic linkages of amylose to yield dextrin, maltose or maltotriose.α (1-4) glycosidic linkagesα (1-4) glycosidic linkages

20. Clinical significance of Amylases Assays for amylase activity in serum and urine are largely of use for: Assays for amylase activity in serum and urine are largely of use for: – Investigation of pancreatic function – Diagnosis of diseases of the pancreas – Detecting the development of complications of pancreatic diseases – Diagnosis of salivary glands diseases and other nonpancreatic disorders. – Increased blood amylase levels in humans are found in: Salivary glands trauma, Mumps, Pancreatitis, Renal failure MumpsPancreatitisRenal failureMumpsPancreatitisRenal failure