Saliva

Effects of acid on apatite structure

"new" enamel contains an apatite high in carbonate which is soluble at a relatively high pH. As the acid reaches this carbonate rich enamel it dissolves, releasing calcium, phosphate, and carbonate into the environment. During this process the pH will increase and the calcium/phosphate concentrations increase resulting in reformation of apatite crystals which will not contain significant amounts of carbonate. This demineralization remineralization cycle occurs continuously throughout the day

factors affecting saliva flow rates

1. (strongest) the mechanical act of chewing, followed by gustatory stimulation with acid the most effecting and sweet the least effective of the sensations. Olfactory sensation has a very weak stimulatory effect. 2. nausea is a strong stimulant of salivary flow 3. other components, such as psychic and learned behaviors have variable strengths and effects 4. medications and drugs can also have a significant effect, particularly those which decrease salivary flow

the buffering capacity of saliva is crucial because saliva must be kept above a pH of

5.5 so the mineralized components of teeth do not dissolve. In whole saliva, quite a bit of acid must be added before the pH drops below the critical level. If bicarbonate is removed however the pH drops off much quicker because only phosphate and urea are acting as a buffer. This shows that *bicarbonate* is the main buffer in *stimulated* saliva. Salivary proteins offer pretty much no buffering capacity. *Phosphate* is the major buffer in *unstimulated* saliva but when salivary flow is stimulated the flow rate increases by 10 fold and the [bicarbonate] goes way up while [phosphate] decreases mainly due to dilution

during metabolism bacteria produce and release acids (formic, acetic, propionic, lactic acids)...

As these diffuse some reach the tooth surface where they produce a drop in pH. Once the pH drops below the point where the solution is no longer saturated with calcium/phosphate with respect to the apatite crystals present, the crystal dissolves.

In an acidic environment bicarbonate will form carbonic acid(pKa is 6.35) which in an open environment will decompose to CO2 and H2O. This eventually removes the hydronium ion from the solution raising the pH and is driven in that direction by the removal of CO2 from the system. The enzyme carbonic anhydrase can significantly increase the rate of this reaction. The parotid and submandibular gland cells produce two CA enzymes:

CA II is a cytosolic enzyme used in the formation of bicarbonate for secretion in saliva. CA IV is the only known CA which is secreted. Its presence in saliva aids in favoring the formation of carbonic acid from bicarbonate in saliva and plaque. *Low levels of salivary CA IV are linked to increased caries risk and DMFT*

mucins(MG1 and MG2)

MG1 is a high molecular weight oligomer while MG2 remains a small monomeric structure. MG2 has significantly more carbohydrates attached per unit of protein than MG1. It plays a role in lubricants, tissue covering, and bacterial clearance.

lactoperoxidase/thiocyanate

SCN- + H2O2--peroxidase--->OSCN- (hypothiocyanate)+ H2O thought to damage bacteria by oxidizing cysteine residues, initially damaging cell membrane making it "leaky". May also enter the cell and inactivate enzymes in a similar manner

statherin

a 43 AA peptide containing two phosphoserine residues near the amino terminus which functions to prevent precipitation of calcium salts from saliva. It is one of the early pellicle forming proteins

proline-rich proteins (PRPs)

a family of proteins characterized by high contents of proline and glycine. Some are basic while others are acidic and at least one is a glycoprotein. PRPs are the major proteins produced by the parotid gland and to lesser extend by other two. It is coded for by 6 genes, over 20 present in saliva. They appear to have a variety of functions including pellicle formation, binding of tannins, calcium binding, and antimicrobial activity.

salivary agglutinin

a large glycoprotein which is identical to glycoprotein gp-340 found in the lungs. It was originally identified by its binding to S. mutans. It functions as a bacterial agglutinin and when bound to the tooth surface acts as a binding site for S. mutans and S. sanguis

The tooth exposed to the oral environment is covered by a complex and changing biofilm. A clean tooth surface is within minutes covered by

a pellicle layer composed of salivary proteins(mostly acidic PRPs and statherin). This layer serves as a diffusion barrier providing limited protection from exposure to the changing oral environment. The PRPs and statherin, as calcium binding proteins, also help maintain the microenvironment saturated with respect to calcium/phosphate without allowing precipitation of calcium phosphate salts. Soon after the formation of the pellicle layer bacteria begin to attach to the layer. The bacteria produce extracellular structures composed of dextrans, levans, and mutans which allow colonization by other bacterial species. As these structures age the organisms change in response to changes produced by bacterial metabolism leading to changes in the organisms present and the structure over time.

cystatins

a superfamily of cysteine protease inhibitors. Both caspases and papain classes of proteases are of the cysteine type. The papain type is produced by many microorganisms. Salivary cystatin acts by binding adjacent of the active site of the enzyme, blocking the active site from interacting with the substrate protein. Cystatins are found in most cells and body fluids.

influencing factors on demineralization/remineralization cycle

amount of acid produced frequency of acid production rate of acid neutralization concentration of calcium and phosphate presence of fluoride ion

fructans (levans)

are primarily beta-2,1 or 2,6 linked. They appear to aid in water retention in the plaque.

lactoferrin

as a member of the non-heme iron binding family of proteins the original role as an antibacterial compound was assumed to be due to complexing iron which is needed for bacterial growth. This is still the mechanism of function in vivo studies. in vitro studies have also shown it to have antibacterial and antiviral properties not reversible by the addition of iron. The amino terminal end is cationic and appears to bind to the organism blocking some required functions.

more recently hCAP-18/LL-37 have been shown to be over expressed in

breast, lung, and ovarian cancer and to stimulate proliferation, migration, and invasion of cancer cells.

salivary amylase

cleaves internal alpha-1,4 bonds in starch/glycogen. It is the most active with large molecules. It is completely inactive at pH below 4. It may help clear oral flora

tannins

consisting of glucose with 5 gallic acids attached to it. This is found in tea and coffee. Tannins bind strongly to proteins in the diet essentially making them indigestible. This is important because many cereal grains and other plant foods produce tannins to protect themselves from insects. When eaten by humans the nutrient value of these plants is greatly reduced. Rats fed a high tannin diet initally show signs of protein deficiency but once the production of basic PRPs is stimulated their symptoms decrease dramatically. Long term tannin consumption has been associated with the development of cancer.

Parotid gland secretes primarily

digestive enzymes(primarily amylase) and protective elements. PRPs(proline-rich proteins) are the primary proteins produced by the parotid gland in humans.

EP-GP(GCDFP-15 in other parts of the body)

extra parotid glycoprotein has been isolated from saliva and other bodily fluids. It appears to function as a bacterial agglutinin binding Streptococcus salivarius, S. oralis, and several other species.

dextrans are produced by

lactic acid producing bacteria, with sucrose as a good source. They are formed enzymatically in the extracellular space. Linkages are primarily alpha-1,6 with alpha-1,3 linkages at branch points with over 1000 glucose units per polymer.

Functions of Saliva

lubrication digestion clearance antimicrobial tissue coating buffering remineralization

enzymatic antibacterials

lysozyme breaks down cell wall of gram + bacteria by hydrolyzing the beta-1, 4 linkage between GlcNAc and N-acetylmuramic acid in peptidoglycans

submandibular glands secrete

mucins which are primarily a lubricant and also contains serous secreting cells sublingual gland is almost purely mucin secretion

composition of saliva

organic: protein, glucose, urea, lipid inorganic: sodium, potassium, calcium, chloride, bicarbonate, and phosphate

Acidic PRPs

rich in acidic residues(glu and asp) with several phosphoserines at the amino terminal end. It may be the initial protein bound during pellicle formation. Along with other pellicle proteins it forms a diffusion barrier which protects the enamel surface. It has been shown to crosslink with statherin through the action of transglutaminase produced by epithelial cells. Like statherin, bind calcium and apatite crystals which helps control calcium salt precipitation

tonicity of saliva

saliva as it is produced in the acini of the salivary glands is isotonic but as it flows through the duct system, to the oral cavity it is extensively modified becoming hypotonic, especially unstimulated saliva the hypotonicity of saliva has several advantages: 1) it increases the hydration of salivary mucins coating the soft tissues 2)low levels of sodium, glucose, urea, and bicarbonate enhance the perception of taste

lingual lipase

secreted by minor salivary glands, most active on short chain FA on the sn-3 position producing a DAG and a free FA. It is relatively hydrophobic partially entering lipid droplets, emulsification not needed. It is not readily inactivated by conditions in the stomach where it continues to act

basic PRP

share most of the structural features of the acidic PRPs except the amino terminal portion (contain lys and arg). They do not bind significantly as part of the pellicle layer. They may play a protective role, particularly in binding tannins and other plant polyphenolic compounds. Basic PRPs are constitutively expressed in humans but are inducible in other animals.

cathelicidin (LL-37)

similar secretion pattern to defensins but they do not loose their antimicrobial activity with increasing ionic strength. The human form is initially 170 AA in length, referred to as human cationic antimicrobial protein 18 or hCAP-18, with the antimicrobial activity located int he 37AA at the carboxy terminal, referred to as LL-37. The amino terminal is homologous with the cysteine antiproteases. Mechanism appears to be the same as defensins with the presence of beta-pleated sheet region similar to that found in defensins. LL-37 is the active antimicrobial peptide and has also been shown to have an immunomodulator to trigger apoptosis and aid in wound healing.

histatins

small histidine rich peptides formed by alternate cleavage of initial translation product. They comprise a little over 2% of the total salivary protein concentration. The role is primarily antifungal; recently shown to keep potassium channel open in C. albicans and in case of histatin 5, to bind to the mitochondria causing an increase in ROS generation, they may also inhibit MMP and have antibacterial properties

Glyco-PRP

these have been shown to behave much like mucins in lubricating the oral cavity and binding microorganisms to clear them from the oral cavity.

saliva flow rate

unstimulated flow, while awake, has an accepted range of 0.1 to 0.5 ml/min with 0.3 ml/min being an accepted norm sleeping flow rate is less than 1% of the waking unstimulated or essentially zero stimulated flow is more variable between individuals ranging from 1 to 7 ml/min and accounts for 80% or more of a persons total daily flow

mutans

which contain alpha-1,4 branches in addition to 1,3 branches are formed by S. mutans under some growth conditions. The mutans tend to be more adherent to the tooth surface than dextrans

defensins

widely found in nature as antimicrobial pepties and are strongly cationic. Secreted by host defense cells, primarily alpha-defensins, and by epithelial cells, primarily beta-defensins. The beta defensin tends to be smaller than alpha. Alpha defensins have a preponderance of arg over lys as the major cationic AA with the, with the six human forms having only arg. Human beta defensins conversely have predominately lys. their strongly cationic nature allows them to easily bind to the anionic bacterial membrane causing leakage across the bacterial membrane and are known to be chemotactic . Down side is that they are susceptible to changes in ionic environment.