1. Biochemistry oral fluid Author - Associate Professor, Department of Biochemistry, E.A. Ryskina

2. Saliva one of the major body fluids There is a biological fluid in the mouth, which is called the mixed saliva or oral fluid (saliva in the future in this lection) and produced by the salivary glands. Saliva contains inorganic and organic components. The inorganic component of saliva is represented by macro and micronutrients which predominantly found in ionized form or bound, such as proteins. Inorganic component: calcium, phosphates, chlorides, sulfates, etc.

3. The chemical composition of saliva saliva Dry residue Organic substances Inorganic substances chlorides sulfates bicarbonates fluoro phosphates proteins albumins globulins Free amino acids Non-protein nitrogen- containing compounds urea creatine ammonia bactericidal enzymes enzymes lysozymes Alpha-amylase

4. Organic components of saliva Organic components comprise 0,8-6,0 grams/liter in the saliva, which is 10-15 times lower than in blood. This components fall in saliva from different sources: -s-salivary glands (in figure); -c-cells of the oral mucosa; -g-gingival fluid (leukocytes); -b-blood; -m-microbial cells. Their number depend on the condition of the oral cavity and the whole organism.

5. The organic composition of saliva Saliva contains proteins, lipids, vitamins, hormones, organic acids, non-protein nitrogenous compounds - urea, uric acid, free amino acids and nucleic acids. Organic substances of saliva can be divided into 2 groups: protein and non-protein nature. Most salivary proteins are glycoproteins that provide viscosity saliva. Glycoproteins composed of a protein part (solid line) and carbohydrate part (polysaccharides).

6. Some protein and non-protein substances included in saliva NAMECONCENTRATION Total protein 1,5 – 3,0 g/l mucins 2,5 –2,7 g/l lysozyme 0,18 g/l uric acid 0,03 –0,17 mol/l urea 1,4 –3,0 mol/l ammonia 2,6 mol/l cholesterol 0,08 – 0,39 mmol/l glucose 0,62 –1,56 mmol/l lactate 20 – 40 mg/l

7. Electrophoresis of saliva shows up to 500 different proteins. About 120 - 150 proteins get to the saliva from the salivary glands. They are called secretory proteins.

8. Salivary proteins can be represented by: Polymorphic protein group: - Proline Rich Proteins - Histidine Rich Proteins - gistatines - Tyrosine Rich Proteins - statherines - Сystatins - Mucins - Salivary enzymes (amylase, peroxidase, etc). Some proteins are represented in a single form: - epithelial growth factor, - nerve growth factor, - lactoferrin, etc.

9. Proline Rich Proteins (PRP) These рroteins contain 70-88% amino acid residues proline, glycine and glutamine. PRP constitute up 70% of all secretory proteins and are divided into 3 groups: - Acidic Proline Rich Proteins - Basic Proline Rich Proteins - Glycosylated Proline Rich Proteins

10. Acidic Proline Rich Proteins - are first deposited on the enamel and begin to form a dental pellicle, because they can bind with calcium saliva their negatively charged terminal; - supervise entry of calcium ions and phosphates to the enamel by preventing the demineralization of enamel; - bind numerous oral microbes and accelerate the formation of tooth plaque.

11. Basic Proline Rich Proteins Basic PRP have antibacterial activity, сan interact with the membrane of streptococci, violate its permeability and cause the death of microorganisms. Basic PRP bind tannins that contains in the food, protecting oral mucosa. Tannins are able to bind proteins and polysacharides oral cavity, there by interfere with the functions of these biomolecules.

12. Glycosylated Proline Rich Proteins - Act as a lubricant, covering the mucous membrane of the mouth; - Accelerate the formation of the dental pellicle, bind to bacteria and deposited on the enamel after acid PRP ; - Contribute to the formation of a lump of food. Protein and carbohydrate linked O-glycosidic bonds and N-glycosidic bonds.

13. Histidine Rich Proteins – Histathines (НRP) In these proteins, the histidine content reaches 25%, much of arginine and lysine. Practically there is no amino acid proline. They are involved in the protection of the oral cavity, providing anti-fungal, anti-virus and anti-microbial action. Histathines penetrate in microbial cells and cause their death. Strongly binding to hydroxyapatite enamel involved in the formation of the dental pellicle. Some histathines inhibit crystal growth of hydroxyapatite.

14. Tyrosine Rich Proteins – statherines These glikofosfoproteins contain much amino acid tyrosine. Statherines participate in the formation of the dental pellicle, prevent excessively rapid precipitation of calcium and phosphorus ions to the enamel surface, tying them. Phosphoserine residues bind calcium are at the N- terminal of the statherines. They inhibit the growth of aerobic and anaerobic bacteria with histatines.

15. Cystatins - acidic low molecular mass proteins of the salive Cystatins perform antimicrobial and antiviral function by the inhibition of enzyme activity of cysteine proteases that hydrolyze proteins of the oral cavity. They inhibit the activity of cysteine proteases by specific binding in the active site of the enzyme with cysteine residues. Cystatins include cathepsins B, H, L, ets. Dental pellicle contains one of cystatins. Cysteine proteases can hydrolyze proteins of saliva when reducing the saliva’s pH.

16. Mucins (from latin. mucus) These glycoproteins contain many amino acid residues proline, serine and threonine and carbohydrate chains of polysaccharides (50-70%). The amino acid residue proline is about 50% of all amino acids included in the mucins. Short polysaccharide chains are attached to serine and threonine O- glycosidic bond. The amino acid proline residues cause the bends of the polypeptide chain mucins. Saliva present mucin-1 (Mr 250 kDa - kilodaltons), and mucin-2 (Mr 100 kDa).

17. Mucins perform specific functions Mucins are involved in the formation of micelles of saliva, due to its ability to bind water. They providing viscosity of saliva. Mucins with proline-rich proteins form dental pellicle that protects cells from oral bacterial, viral, chemical action. Mucins act like a lubricant not just in the oral cavity, but also in the intestines and bronchi.

18. Molecular structure of mucin Mucin consists of two parts – a protein (solid line) and carbohydrate parts. Carbohydrate part consists of short chains of polysaccharide. Fucose, galactose, glucose, N- acetylglucosamine, and other sugar are included in polysaccharide chains.

19. Saliva enzymes More than 100 enzymes are active in the saliva. Basically, enzymes are synthesized by the salivary glands, part falls into the saliva from the destroyed epithelial cells, bacterial cells or from the blood. Saliva contains the following enzymes: - proteases (cathepsins A, B, H and L) - phosphatases - glycosidases - DNase and RNase - enzymes - antioxidants, etc.

20. Glycosidase enzymes of saliva They include: - digestive saliva enzymes - maltase, lipase, a– amylase and other. Salivary a-amylase can hydrolyze 1-4 bond in starch and glycogen food, can destroy polysaccharides of the membrane gonococci, showing the protective action. - bacterial enzymes: β - glucuronidase, neuraminidase, hyaluronidase. - antibacterial enzyme – lysozyme, which can destroy the walls of bacteria. Bacterial enzymes can be activated by acidification at pH of saliva which can lead to the destruction of tooth tissue. For example: mucins can be cleaved by β - glucuronidase, which leads to the development of gingivitis and caries.

21. Lysozyme an antibacterial enzyme Lysozyme is a glycoprotein (molecular mass of 15-17 kDa), containing up to 50% of the carbohydrate components. This enzyme can hydrolyse 1,4- glycosidic bonds between N- acetylglucosamine and N- acetylmuramic acid. N-acetylmuramic acid is a polysaccharide cell walls of bacteria.

22. DNase and RNase DNase and RNase disrupt nucleic acid bacteria and viruses and thus exhibit antiviral and antimicrobial effect.

23. Enzymes - Antioxidants A large amount of reactive oxygen species (ROS) have a devastating effect on the components of the cell membranes of the oral tissues. Protective action have enzymes which reduce the concentration of free radicals: - Superoxide dismutase (SOD) - catalase - glutathione peroxidase et al.

24. Lactoferrin - iron-binding protein Lactoferrin binds iron ions are essential for bacterial growth, and thus provides an antibacterial effect. Lactoferrin is able to interact directly with the polysaccharides of the membrane Escherihia coli and cause their death. Lactoferrin plays an important role in maintaining immunity of newborns.

25. Salivary glands are synthesized by biologically active substances (BAS) saliva: Epithelial growth factor (EGF) - enhances bone resorption (breakdown) of bone tissue and division odontoblasts. Nerve growth factor (NGF) – has a powerful anti-inflammatory effect. Parotin - promotes mineralization. Renin - vasoconstrictor activity. Biologically active substances of saliva have endocrine function and are involved in the regulation of homeostasis of many organs and tissues of the body.

26. Immunoglobulins saliva - factors of nonspecific protection Saliva contains all five immunoglobulin classes and secretory - IgAs, produced by the salivary glands. The main function of secretory IgAs is the inhibition of attachment of bacteria on the mucosal surfaces of the oral cavity. IgAs has bactericidal, antiviral and antitoxic action.

27. Structure IgAs IgAs is in connection with the S- glycoprotein (secretory component), which protects it from degradation by enzymes.

28. Protective properties of saliva The protective function of saliva is carried out due to the presence in its composition: Protective proteins (mucins, PRP, histatines, cystatins, statherines, laktofferin et al.) leukocytes (lysosomal enzymes) Immunoglobulins (IgA, IgE, IgD, IgM, IgG, and particularly secretory - IgAs) Enzymes (lysozyme, superoxide dismutase, catalase, glutathione peroxidase, DNAse and RNAse, a-amylase)

30. Inorganic substances in saliva mmol/l (T.P. Vavilovа) substancesalivablood plasma Na +6,6 - 24130 - 150 K+12 - 253,6 - 5,0 Cl -11 - 2097 - 108 Ca2+ 0,75 – 3,02,1 – 2,8 Pi 2,2 – 6,51,0 – 1,6 Pi total 3,0 – 7,03,0 – 5,0 НСО3-20 - 6025 SCN- 0,5 – 1,20,1 – 0,2 Сu2+0,30,1 I-0,10,01 F- 0,001 – 0,15 0,15

31. Calcium and phosphates The content of calcium ions in the saliva is in the range of 0,75 – 3,0 mmol/l (as in plasma). Calcium can be present in ionized (Ca2 +) or related with protein forms. Phosphates are in the saliva in the form of free ions hydrogenphosphate and dihydrogenphosphate, which accounts for 70 - 95% of the total phosphate. There is more phosphate content in saliva than in blood.

32. The functions of some ions of saliva Ions Na + and K + with other ions determine the osmotic pressure, buffer capacity and stability of the micelles saliva. Bicarbonates are components of the buffer system saliva. The fluoride ions get from gingival sulcus to the saliva. Fluoro accelerates remineralization and has an inhibitory effect on bacterial growth.

33. Micellar structure of saliva is the basis of mineralizing function of saliva. Saliva is supersaturated with calcium and phosphate ions, but it does not lead to deposition of these minerals on the tooth surface. This is prevented by the micelle structure of saliva. Micelles - colloid formation (structural unit saliva) supporting the calcium salt in a dissolved state. The figure shows the crystal structure of saliva.

34. The structure of the micelles The core of the micelles is insoluble calcium phosphate Ca 3 (PO 4 ) 2. Are arranged around a core of calcium ions, calcium hydrogenphosphate, calcium dihydrogenphosphate and protein molecules. The major proteins are mucins and statherines (in the figure they are depicted shown in circles and ovals).

35. Оver the tooth surface formation Throughout the human life on the enamel surface can be formed dental pelicula and tooth plaque. Mineralization of tooth plaque leads to the formation odontolith.

36. Оn the surface of the tooth is supported by the constancy of the pH The constancy of pH buffer systems is provided by saliva. Sealing or thickening plaque deprives saliva opportunities to exercise their protective effect.

37. Depending on the nature of food and microorganisms in tooth plaque can be realized two opposite situations: 1. Formed acidic environment (its formation promotes of foods rich in carbohydrates), in which the enamel demineralization and caries. 2. Formed alkaline environment (in it high concentrations of calcium and phosphate accumulates), the conditions for the precipitation of calcium salts and the formation of odontolith.

38. Currently, dental caries is associated with a local change in pH on the surface of a tooth plaque due to enzymatic degradation of carbohydrates (glycolysis), carried out by microorganisms and the formation of organic acids. The interaction of these factors determines the appearance of foci of demineralization.

39. Gingival fluid - biological oral fluid, which bathes the gingival sulcus Gingival fluid includes lowered epithelial cells, white blood cells (the main source of income in the saliva), microorganisms, electrolytes, protein components and enzymes. There is a close interconnection between the degree of increase of inflammatory changes in the periodontium and the level of activity of hydrolytic enzymes of leukocytes.

40. The most important enzymes leukocytes gingival fluid, which has a protective effect on periodontal tissues: Acid phosphatase (a marker of lysosomes); Alkaline phosphatase; Various glycosidases; Proteases (cathepsins B, elastase, collagenase); Lysozyme; Phospholipase; Myeloperoxidase et al.

41. Function lysosomal’s enzymes of leukocytes These enzymes increase the permeability of capillaries and therefore facilitate further release of leukocytes. Lysosomal’s enzymes attacks bacteria and destroy the whole bacterial cell. For example: phospholipase, lysozyme. Alkaline phosphatase is necessary to perform the functions of phagocytic leukocytes.

42. Myeloperoxidase of leukocytes is involved in the formation reaction of hypochlorite (bactericidal effect) Leukocyte myeloperoxidase catalyzes the reaction: H 2 O 2 + Cl‾→ H 2 O + OCl‾ Hypochlorite (OCl‾), that produced in the reaction, has more potent bactericidal effect than hydrogen peroxide in ten times.

43. Thank you for your attention!