Saliva

Saliva is the watery and usually frothy substance produced in the mouths of humans and some animals. In animals, saliva is produced in and secreted from the salivary glands.

Functions

Digestion

The digestive functions of saliva include moistening food, and helping to create a food bolus, so it can be swallowed easily. Saliva contains the enzyme amylase that breaks some starches down into maltose and dextrin. Thus, digestion of food occurs within the mouth, even before food reaches the stomach. Salivary glands also secrete enzyme to start fat digestion. This is useful for infants to digest the fat in milk.

Role in emesis

The importance of the salivary protective function can be demonstrated by considering a scenario where an individual is about to vomit. Vomit contains gastric substances which are extremely acidic and will erode teeth. A protective reflex occurs before the individual prepares to vomit. Signals are sent from the brain to the salivary glands via the involuntary nervous system to cause increased saliva secretion, even before vomiting occurs. Thus, when vomiting does occur, there is already saliva present in the mouth acting to minimize the acidity and thus prevent destruction of tooth structure.

Pellicle deposits

In addition to this, saliva is responsible for depositing salivary pellicle that covers the entirety of the tooth surfaces. This pellicle is believed to play a role in plaque formation, though there is evidence that it may also act as a protective barrier between acids and the tooth surface.^[1]

Disinfectants

A common belief is that saliva contained in the mouth has natural disinfectants, which leads people to believe it is beneficial to "lick their wounds". Researchers at the University of Florida at Gainesville have discovered a protein called nerve growth factor (NGF) in the saliva of mice. Wounds doused with NGF healed twice as fast as untreated and unlicked wounds; therefore, saliva does have some curative powers in some species. NGF has not been found in human saliva; however, researchers find human saliva contains such antibacterial agents as secretory IgA, lactoferrin, and lactoperoxidase.^[2] It has not been shown that human licking of wounds disinfects them, but licking is likely to help clean the wound by removing larger contaminants such as dirt and may help to directly remove infective bodies by brushing them away. Therefore, licking would be a way of washing, useful if purer water isn't available to the animal or person.

The mouth of animals is the habitat of many bacteria, some of which may be pathogenic. Animal (including human) bites are routinely treated with systemic antibiotics because of the risk of septicemia.

Stimulation

The production of saliva is stimulated both by the sympathetic nervous system and the parasympathetic.^[3]

The saliva stimulated by sympathetic innervation is thicker, and saliva stimulated parasympathetically is more watery.

Daily salivary output

There has been some disagreement regarding the daily salivary output in a healthy individual. Today, it is believed that the average person produces approximately 700mL of saliva per day, which is much less than was once thought.

Contents

Produced in salivary glands, saliva is 98% water, but it contains many important substances, including electrolytes, mucus, antibacterial compounds and various enzymes. ^[4]

It is a fluid containing:

• Water
• Electrolytes:
  • 2-21 mmol/L sodium (lower than blood plasma)
  • 10-36 mmol/L potassium (higher than plasma)
  • 1.2-2.8 mmol/L calcium
  • 0.08-0.5 mmol/L magnesium
  • 5-40 mmol/L chloride (lower than plasma)
  • 25 mmol/L bicarbonate (higher than plasma)
  • 1.4-39 mmol/L phosphate
• Mucus. Mucus in saliva mainly consists of mucopolysaccharides and glycoproteins;
• Antibacterial compounds (thiocyanate, hydrogen peroxide, and secretory immunoglobulin A)
• Various enzymes. There are three major enzymes found in saliva.
  • α-amylase (EC3.2.1.1). Amylase starts the digestion of starch and lipase fat before the food is even swallowed. It has a pH optima of 7.4.
  • lysozyme (EC3.2.1.17). Lysozyme acts to lyse bacteria.
  • lingual lipase (EC3.1.1.3). Lingual lipase has a pH optimum ~4.0 so it is not activated till entering an acidic environment.
  • Minor enzymes include salivary acid phosphatases A+B (EC3.1.3.2), N-acetylmuramyl-L-alanine amidase (EC3.5.1.28), NAD(P)H dehydrogenase-quinone (EC1.6.99.2), salivary lactoperoxidase (EC1.11.1.7), superoxide dismutase (EC1.15.1.1), glutathione transferase (EC2.5.1.18), class 3 aldehyde dehydrogenase (EC1.2.1.3), glucose-6-phosphate isomerase (EC5.3.1.9), and tissue kallikrein (EC3.4.21.35).
• Cells: Possibly as much as 8 million human and 500 million bacterial cells per mL. The presence of bacterial products (small organic acids, amines, and thiols) causes saliva to sometimes exhibit foul odor.

Salivary Buffering

Acid containing beverages and food are a menace to teeth as these agents contribute to erosion of tooth surfaces (1,2). Enamel and dentine are composed primarily of a carbonate substituted calcium deficient hydroxyapatite Ca10(PO4)6(OH)2. When hydroxyapatite is in contact with water (saliva), the following reaction takes place (3):

Reaction 1: (Solid) Ca₁₀(PO₄)₆(OH)₂ ⇌ 10Ca²⁺ + 6PO₄3– + 2OH^- (Solution)

Addition of acid to this solution e.g. while drinking apple juice (pH 3), leads to a shift of the chemical equilibrium to the dissolution side as hydroxyl ions (OH–) are removed from the tooth. Dissolution ends and remineralisation of dental hard tissue occurs when the pH in close proximity of the tooth is rising (2). Saliva that permanently covers the structures forming the oral cavity contains three buffer systems, the carbonate, the phosphate and the protein buffering system (4,5). Together these buffer systems form the first line of defence against acidic or basic challenges, a salivary function of utmost importance. Inorganic buffers are aqueous solution of acids and their conjugated bases that are resistant to pH changes upon the addition of small amounts of acid or base (6). A buffer, as defined by Van Slyke is "a substance which by its presence in solution increases the amount of acid or alkali that must be added to cause unit change in pH” (7). This attribute of the buffering solution is quantified by the buffering value β which is calculated as the quotient of the differential addition of acid over differential pH change(7). The primary salivary buffer is composed of carbonic-acid (H₂CO₃) and hydrogencarbonate also known as bicarbonate (HCO₃^-). Bicarbonate is excreted by the submaxillary duct system by means of an active transport mechanism (8). A bicarbonate containing solution, like saliva exhibits optimal buffering when its pH is equal to the negative logarithm of the acidic constant (pka) of carbonic acid, which is 6.1 (37°C, (9)). According to the Henderson-Hasselbach equation (10), the solution has a maximal buffer range from 5.1 to 7.1 (pka +/- 1 pH unit, (6)) Beyond this range, no buffering from the carbonate system occurs. The reactions of the carbonate system with acids and bases are (4):

Displacement of carbon dioxide from hydrogencarbonate

Reaction 2: HCO₃^- + H₃O⁺ ⇌ H₂CO₃ + H₂O

Reaction 3: H₂CO₃ ---> CO₂ + H₂O

Phase transfer of carbon dioxide

Reaction 4: CO₂(aq) ⇌ CO₂(g)

Dissociation of carbonic acid

Reaction 5: H₂CO₃ + H₂O ⇌ HCO_3- + H₃O⁺ (1st step)

Reaction 6: HCO₃^- + H₂O ⇌ CO₃2- + H₃O⁺(2nd step)

After addition of base:

Reaction 7: HCO^3- + OH- ⇌ CO₃^2- + H₂O

Reaction 8: CO₃^2- + 2H₃O+ --> H₂CO₃ --> Reaction 3+4

The concentration of hydrogencarbonate ranges from 5 mM in resting saliva (4) up to 60 mM in stimulated saliva (8). Upon acidic or basic challenges, the carbonate system forms carbonic acid, which rapidly decays to form water and gaseous carbon dioxide. This feature of the carbonate system is called phase-buffering (4). Buffering will occur as long as no more than 50% of the hydrogencarbonate is transformed into carbonic acid. The amount of acid that is buffered is called the ‘buffer power B’ or ‘molecular buffer bM’ with unit mol/l (7). The buffer power is calculated according to the formula B=c2/2c=0.5c, were c is the concentration in mol/l of the buffer components (7). The secondary salivary buffer is based on the inorganic ions di-hydrogenphosphate (H2PO4-) and mono-hydrogenphosphate (HPO₄^2-). Since the pH of saliva varies between 5.8 and 8 (11), the second dissociation constant of phosphoric acid pka=7.1 defines the buffering optimum of the phosphate system at pH 7.1 (4,8) and a buffer range from pH 6.1 to 8.1 (Fig. 1). The concentration of di-hydrogenphosphate in resting saliva is 7.8 mM and less than 1 mM in stimulated saliva (8). The concentration of mono-hydrogenphosphate varies from a level below 1 mM in resting saliva to 3 mM in stimulated saliva (8). The total phosphate concentration in resting saliva is 8.4 (+/-) 3 mM (12). The reactions of the phosphate system with acids and bases are (4):

Reaction 9: HPO₄2- + H₃O+ ⇌ H₂PO₄- + H₂O (addition of acid)

Reaction 10: H₂PO₄- + OH- ⇌ HPO₄^2- + H₂O (addition of base)

The buffering levels of the carbonate system and the phosphate system have an overlapping zone from pH 6.1 to 7.1 where both systems synchronously are active. Further information and an in-depth discussion of the carbonate and phosphate buffer systems were published by Bardow and co-workers (4). As high concentrations of inorganic salts such as hydrogencarbonate and di-hydrogenphosphate might interfere with biological reactions, the human body has found another way to provide additional buffering. Similar to the zwitterionic Good-buffers (13,14), proteins have anionic and cationic sites present as non-interacting carboxylate and ammonium side chains. These types of buffers display good water solubility and have a low interference with biological processes. The protein buffer system which is part of the human salivary proteome, currently, is known to comprise 944 protein species (15-22). There is insufficient evidence to known how many of the 944 components of the salivary proteome substantially contribute to buffering. As proteins have many acidic and basic side chains no discrete pka value exists to estimate their buffer level. The amino acids in a protein that can be ionized are referred to as titratable groups. They are divided into acidic and alcaline residues. Aspartic acid (Asp), Glutamic acid (Glu), Cysteine (Cys), Serine (Ser), Tyrosine (Tyr), Tryptophane (Thr) belong to the acidic residues as well as the C-terminal end of the protein. Alcaline residues are the Histidine (His), Lysine (Lys) and Arginine (Arg) and the N-terminal end. For the acidic groups glutamic- and aspartic acid (Asp pka = 4,4 and Glu pka =3.9), the following reaction takes place at a pH above pH 4.4.

Reaction 11: (Asp), R-CH₂-COOH ⇌ R-CH₂-COO^- + H⁺

Reaction 12: (Glu), R-(CH₂)₂-COOH ⇌ R-CH₂-COO^- + H⁺

R stands for the rest of the Amino acid (H₂N-CH-COOH).

When the pH is higher than the pka the equilibrium of reaction 9 and 10 is on the left side. When acid is added and the pH reaches a value below the pka the equilibrium of both reactions is shifted to the right side. For the basic groups lysine (pka = 10.5) and arginine (pka = 13.2), the following reactions take place:

Reaction 13: (Lys), R-(CH₂)₄-NH₂ ⇌ R-(CH₂)4-NH- + H+

Reaction 14: (Arg), R-(CH₂)₃-NH-C(=NH)-NH₂) ⇌ (R-(CH₂)3-NH-C(=NH)-NH)+ H+

At a pH lower than the pka the equilibrium of reactions 13 and 14 is on the right side. As soon as the pH is higher than the pka the equilibrium of reactions 11 and 12 is shifted to the left side. Being part of protein titrable groups are in a complex environment produced by the three dimensional structure of the protein. This can profoundly affect the pka and the reaction of each acidic or basic group. As the pKa of a titrable group is determined by its micro-environment, it can take on a range of values radically different from those measured for individual amino acids. The estimation of individual pka values of a titrable group in a protein is done for instance by the Poisson-Boltzmann method (23). Based on such data titration curves can be calculated to estimate isoelectric points of proteins (24). The isoelectric point which is the pH where the sum of all positive and negative charges equals zero, is used to define the pH of optimal buffering. Of 944 proteins, 346 salivary proteins have an isoelectric point beyond the buffer range of the carbonate and phosphate system (74 below pH 5.1 and 272 above pH 8.1). Therefore buffering based on protein can be awaited above pH 8.1 and below pH 5.1. Carbonic anhydrases catalyze the reversible hydration of carbon dioxide (5). They therefore deserve special consideration here. Carbonic anhydrases are located in the human alimentary tract (25,26) and participate in the maintenance of pH homeostasis, in biological fluids of the human body. Of 11 isoenzymes, carbonic anhydrase II and IV are involved in oral physiology (27). Carbonic anhydrase IV is located in the enamel pellicle and not involved in the regulation of actual salivary pH or buffer capacity (5). Carbonic anhydrase II is thought to produce bicarbonate in saliva but until recently, it had not been determined if, and how much, bicarbonate was produced (5). With the exception of the total protein concentration from 1.5 to 6.5 g/l (28) and the presence of 944 different protein species in saliva, the information about the protein buffer system is scarce (29-33). Buffering from protein in saliva is likely to occur as in the human body proteins are the most potent buffering substances (34). More detailed Information about protein buffering in human saliva can be found at PLoS One [1] or the saliva homepage [2]. A method to visualize salivary proteins after electrophoresis can be found at the Ruthenium homepage [3] or at Ruthenium(II) tris(bathophenanthroline disulfonate).

1.Zero D. Caries Res 2004; 38 Suppl 1: 34-44.

2.Zero D. Martin Dunitz Ltd, 2000:121-139.

3.Dawes C. J Can Dent Assoc 2003; 69: 722-724.

4.Bardow A, Moe D, Nyvad B, Nauntofte B. Arch Oral Biol 2000; 45: 1-12.

5.Lenander-Lumikari M, Loimaranta V. Adv Dent Res 2000; 14: 40-47.

6.Mortimer CE. Säuren und Basen. In, Das Basiswissen der Chemie. 5 ed. Stuttgard: Georg ThiemeVerlag; 1987:283

7.Van Slyke D. J Biol Chem 1922; 52: 525-570.

8.Kreusser W, Hennemann H, Heidland A. Verh Dtsch Ges Inn Med 1972; 78: 1518-1522.

9.Sigaard-Anderson O. In, The acid-base status of the blood. Copenhagen: Munksgaard; 1963

10.Hasselbach KA. Biochem Z 1917; lxxvi: 112-143.

11.Larsen MJ, Jensen AF, Madsen DM, Pearce EI. Arch Oral Biol 1999; 44: 111-117.

12.Rehak NN, Cecco SA, Csako G. Clin Chem Lab Med 2000; 38: 335-343.

13.Good NE, Winget GD, Winter W, et al. Biochemistry 1966; 5: 467-477.

14.Good NE, Izawa S. Hydrogen ion buffers. Methods Enzymol 1972; 24: 53-68.

15.Kojima T, Andersen E, Sanchez JC, et al. J Dent Res 2000; 79: 740-747.

16.Ghafouri B, Tagesson C, Lindahl M. Proteomics 2003; 3: 1003-1015.

17.Yao Y, Berg EA, Costello CE, Troxler RF, Oppenheim FG. J Biol Chem 2003; 278: 5300-5308.

18.Huang CM. Arch Oral Biol 2004; 49: 951-962.

19.Vitorino R, Lobo MJ, Ferrer-Correira AJ, et al. Proteomics 2004; 4: 1109-1115.

20.Wilmarth PA, Riviere MA, Rustvold DL, et al. J Proteome Res 2004; 3: 1017-1023.

21.Hu S, Xie Y, Ramachandran P, et al. Proteomics 2005; 5: 1714-1728.

22.Xie H, Rhodus NL, Griffin RJ, Carlis JV, Griffin TJ. Mol Cell Proteomics 2005; 4: 1826-1830.

23.Nielsen JE, Vriend G. Proteins 2001; 43: 403-412.

24.Kuramitsu S, Hamaguchi K. J Biochem (Tokyo) 1980; 87: 1215-1219.

25.Parkkila S, Kaunisto K, Rajaniemi L, et al. J Histochem Cytochem 1990; 38: 941-947.

26.Parkkila S, Parkkila AK, Juvonen T, Rajaniemi H. Gut 1994; 35: 646-650.

27.Kadoya Y, Kuwahara H, Shimazaki M, Ogawa Y, Yagi T. Osaka City Med J 1987; 33: 99-109.

28.Alfonsky D. Drawer: University of Alabama press, 1961.

29.Lilienthal B. J Dent Res 1955; 34: 516-530.

30.Dawes C. J Physiol 1981; 320: 139-148.

31.Freidin LI, Nikolaev AA, Freidin BL. Stomatologiia (Mosk) 1985; 64: 16-17.

32.Van Nieuw Amerongen A, Bolscher JG, Veerman EC. Caries Res 2004; 38: 247-253.

33.Pedersen AM, Bardow A, Nauntofte B. BMC Clin Pathol 2005; 5: 4.

34.Stoelting KR. Pharmacology & Physiology in Anesthetic Practice. Third Edition ed, 1999:702.

See also

• Tongue

References

1. ^ The acquired enamel pellicles in adults and children
2. ^ Discover Magazine, "The Biology of ...Saliva" October 2005
3. ^ Physiology at MCG 6/6ch4/s6ch4_7
4. ^ Physiology at MCG 6/6ch4/s6ch4_6

External links

• MeSH Saliva