Download Biochemistry of saliva and tooth tissues

2017-01-12
Biochemistry of saliva
and tooth tissues
[email protected]
Tooth
is a complex system of specialized tissues
Tooth tissues
enamel
the hardest part of the tooth
acts as a protective tissue that covers the exposed part of
a tooth (the crown)
dentin
contains collagen
is similar in composition to bone
is the tissue that forms the mainly mass of a tooth and
supports the enamel
dental pulp
a soft connective tissue containing nerves and blood
vessels
nourish the tooth
cementum
covers the dentine outside of the root (under the gum
line)
attached to the bone of the jaw with little elastic fibers
1
2017-01-12
Enamel
the hardest substance in the human body
contains the highest percentage of minerals (95% is
hydroxyapatite), water and organic material
initially composed of high amount of protein;
during tooth matures proteins are removed and the voids
are backfilled with HAP
Remineralisation of teeth - repairing damages of the tooth
only to a certain degree
The maintenance and repair of human tooth enamel
- one of the primary concerns of dentistry.
Dentin
is a mineralized connective tissue with an
organic matrix of collagenous proteins
contains exclusively genetic type I collagen
its poilpeptide chain is rich in glycine (Gly),
proline (Pro) and hydroxyproline (Hyp)
composed of mineralized calcium phosphate
(calcium phosphate phase called
hydroxyapatite (HAP) Ca10(PO4)6(OH)2) 70%
- within a matrix of collagen fibrils (20%) and
water (10%)
HAP - living mineral that is continually
grown, dissolved and remodeled in response
to signals of internal (e.g., pregnancy) and
external (e.g., gravity, exercise) environment
Crystalline structure of
hydroxyapatite in enamel
20-50nm Hydroxyapatite Nanoparticles
2
2017-01-12
Cementum and dental pulp
Cementum
is a specialized bony substance covering the root of a tooth
45% inorganic material (mainly hydroxyapatite),
33% organic material (mainly collagen)
22% water
Cementum is excreted by cementoblasts within the root of the
tooth and is thickest at the root apex.
Dental pulp
the part in the center of a tooth made up of living soft tissue and
cells called odontoblasts
commonly called 'the nerve', although it contains many other
structures which are not nerves
Dental plaque
biofilm consisting of large amounts of various
bacteria which forms on teeth
if not removed regularly, can lead to dental
cavities (caries) or periodontal problems (such
as gingivitis)
plaque can mineralize along the gingiva,
forming tartar
microorganisms form the biofilm: almost
entirely bacteria (mainly Streptococcus and
anaerobes)
Certain bacteria in the mouth in the absence of
oxygen produce lactic acid (which dissolves the
calcium and phosphorus in the enamel). This
proces - demineralisation, leads to tooth
destruction.
3
2017-01-12
Dental caries
infectious disease which damages the structures of
teeth
caused by certain types of acid-producing bacteria
which cause the most damage in the presence of
fermentable carbohydrates such as sucrose, fructose,
and glucose
The resulting acidic levels in the mouth affect teeth
because a tooth's special mineral content causes it to
be sensitive to low pH.
Teeth demineralisation:
2+
10
4)6
2
9 2
4) 6
2
optimal pH for remineralization: 6.4-7.8 (pH<6,2
leads to the demineralization )
ph<6,2 leads to the demineralization
Mg2+, Mn2+, Zn2+, Cu2+, Si2+ ions increase
mineralization
4
2017-01-12
Saliva
the most important factor maintaining homeostasis in the oral cavity
Saliva contains:
fluid produced by the salivary glands
gingival crevicular fluid (GCF)
transudate - extravascular fluid with low protein content and a low specific
gravity
exfoliated epithelial cells, white blood cells, buccal cells,
serum proteins,
cell-free DNA, RNA
bacteria and other pathogens
Daily salivary output
During the night: 3 ml/hr.
Resting saliva: 30 ml/hr.
During the meal: 120 ml/ hr.
Under normal physiological conditions: about 1.0 liter per day.
5
2017-01-12
Salivary secretion:
Saliva is secreted by:
salivary glands
parotid gland
sublingual gland and
submandibular gland
small salivary glands scattered in the mucous membrane of
the lips, palate, tongue and cheeks.
Parotid glands secret clear, watery, serous fluid which is rich in
amylase.
Sublingual glands secret primarily mucus and most viscous saliva.
Submandibular glands produce primarily serous fluid with some
mucus.
Salivary secretion:
The first stage primary saliva:
saliva produced in the acinar cells
isotonic to plasma
rich in NaCl
The second stage final saliva:
active absorption of Na +, Ca2 +, Cl- and
HPO42- ions
passive secretion of HCO3- and K+ ions
osmotic gradient causes the passive
transport of water.
Ions resorption faster than secretion, small
water penetration ultimately saliva is
hypotonic
At very high flow: final saliva composition
approaches to the composition of the
priamary saliva.
Secretion of HCO3- anions determines an
increased pH of the saliva flowing through
the ducts.
AQP5 - Aquaporin 5 is a water channel prote, plays a role in the generation of saliva, tears and pulmonary secretions.
6
2017-01-12
Components of saliva:
Water: 99%
Organic components:
enzymatic and non-enzymatic proteins
low molecular nitrogen compounds
lipids
Inorganic components:
cations and anions
Organic compounds proteins:
Mucins:
one of the major proteins of the oral cavity
belong to the class of glycoproteins
increased quantity of mucin in the saliva increases its density and
viscosity
Mucins function:
Tissues coating
sugar residue
protecvive coating about soft and hard tissues,
primary role in formation of acquired pellicle
concentrates anti-microbial molecules at mucosal interference
Lubrification increases lubricating qualities (film strength)
Anti-bacterial:
aggregarion of bacterial cells
bacterial adhesion
polipeptide chain
A sugar component of mucins is sialic acid (N-acetylneuraminic acid).
7
2017-01-12
Organic compounds proteins:
Proline reach proteins, PRPs
inhibitor of calcium phosphate crystal growth
p
(0.1-
pellicle
surface)
Statherins:
prevent precipitation or crystallization of supersaturated calcium phosphate in
ductal saliva and oral fliud
cause demineralization of early carious lesion
lubricant
Organic compounds proteins:
Histatins
small histidine-rich proteins
potent inhibitor of bacteria and Candida albicans growth - histatins molecule
has a positively charged side chain that binds to the negative charges of the
cell membrane phospholipids of microorganism (bacterium or fungus)
it leads to loss of microogranism membrane integrity
various ions and organic compounds (eg. ATP) "run away" from the microbial cell
this results in damage and consequent microorganism cell death
natural metalloproteinase inhibitors (histidins have the ability to bind Zn2+
and Cu2+ ions - activators of metalloproteinases - enzymes that degrade the
extracellular matrix)
remineralization function
8
2017-01-12
Organic compounds proteins:
Cystatins
inhibitor of harmful cysteine-proteases
protective against unwanted proteolysis (bacterial proteases lysed
leukocytes), inhibit proteases in periodontal tissues
inhibit the precipitation of calcium phosphates in the saliva
play an important role in the prevention of periodontal diseases
alleviate inflammation in the oral cavity
occur mainly in submandibular salivary secretions
Lactoferrins
glycoprotein that has the ability to bind Fe3+ ions (some of the oral
bacteria require these ions to growth)
Organic compounds proteins:
Immunoglobins:
IgA
represents 90% of the antibodies in the saliva of
produced by plasma cells in the salivary gland epithelium and the epithelial cells of oral mucosa
affects phagocytosis of streptococci by leukocytes
IgM
is secreted in part by the salivary gland, and a part comes from the tissue fluid
IgG
represent 70% of the antibodies in the gingival fluid
comes to saliva from gingival pockets and intercellular space through inflamed mucosa
IgA and IgG have the ability to agglutinate Streptococcus mutans facilitating their removal from the mouth
with swallowing saliva.
Therefore they influence reducing the adhesion of bacteria to the epithelium of the cheeks and tooth
enamel.
9
2017-01-12
Organic compounds enzymes:
Lysozyme
present in numerous organ and most body fluids
oral lysozyme is derived from at least 4 sources:
minor and major salivary glands
phagocytic cells
gingival crevicular fluid
has anti-microbial activity by inhibition of bacterial adhesion to tooth surface and
inhibition of glucose uptake and acid production.
has muramidase activity (lysis of peptidoglycan layer).
damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between
N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan
and between N-acetyl-D-glucosamine residues in chitodextrins
Organic compounds enzymes:
lpha-amylase
-1,4-glycosidic bonds in the starch
activated by chloride ions
the optimum pH for its action: 6-7
is helpful in removing the carbohydrate fibers located
between teeth
It is responsible for the formation of a glycoprotein complex
of pellicle. This complex is formed directly on the washed
teeth. On the pellicle the plaque (responsible for most of oral
diseases dental caries, periodontitis) is formed. Alfaamylase has high affinity for bacteria and it binds to them.
isoenzyme
of alpha-amylase present in saliva
Increased activity of the enzyme
in the blood can indicate diseases of the salivary
glands or pancreas!
10
2017-01-12
Organic compounds enzymes:
Lingual lipase
secreted by the Von Ebner's glands at the
root of the tongue in the first days after
birth
takes part in the digestion of emulsified
lipids contained in milk
little importance in the digestion of lipids
in other periods of life
Organic compounds antioxidant enzymes:
1. System of salivary peroxidases:
Lactoperoxidase
catalyzes the hydrogen peroxide (H2O2) oxidation of several acceptor molecules:
reduced acceptor (SCN-) + H2O2
oxidized acceptor (OSCN-) + H2O
OSCN ions inhibit the activity of bacterial hexokinase
Source of the hydrogen peroxide usually is the reaction of glucose with oxygen in the presence of the enzyme
glucose oxidase that takes place in saliva.
Lactoperoxidase system is effective in killing of aerobic and certain anaerobic microorganisms.
Myeloperoxidase
released in inflammatory processes
oxidizes (with simultaneous reduction of H2O2) chlorine to hypochlorous which has antiviral and antibacterial
properties
2. Catalase:
capable of defending against reactive oxygen species
decomposes the hydrogen peroxide into water and oxygen: 2 H2O2
2O
+ O2
Saliva is the first line of defence against reactive oxygen species (free radicals).
11
2017-01-12
Non-protein nitrogenous substances
Uric acid
concentration in saliva is 40-240 mmol/L
small molecule antioxidant (approx. 70-80% of the antioxidant activity of
saliva)
Urea
metabolic product of salivary glands
important in the action of the buffer system in saliva - ammonia, which is
derived from urea decomposition, involves (reemoves) an excess of H+ ions
Creatinine, amino acids
may be derived from blood
to saliva are transferred by the salivary glands
Non-protein organic substances
Carbohydrates
most salivary carbohydrates bind to proteins forming glycoproteins
higher concentrations are found in diabetics
Lipids
simple and phospholipids,
cholesterol occur in small amounts in the secretions of the salivary
glands
may be obtained from the cell membranes of the salivary glands
Steroid hormones
their presence and concentration in saliva depends on the
concentration in the blood
Opiorphin
a peptide, compound with analgesic properties
12
2017-01-12
Inorganic components of saliva:
their content in saliva is not constant
come mainly from the blood (with the exception of HCO 3-)
occur in ionic form
CATIONS:
Na+, K+
are involved in the transport of active compounds through cell membranes.
the presence of Na+ in hydoxyapatites increases the solubility of enamel in acids
Na+ regulates salivary osmotic tensiom
Ca2 +
builds hard tissue
involved in the maturation of enamel and remineralization of initial damage
activator of certain enzymes in saliva
Mg 2+
involved in creating the structure of the teeth
an activator of certain enzymes in saliva
content of Mg2+ in the hydroxyapatite increases the solubility of enamel in acid
Inorganic components of saliva:
ANIONS:
Clregulates osmotic tension of saliva
-amylase
Faffects the structure and processes of remineralization of enamel.
antibacterial properties
I
plays a role in the defense mechanisms mainly by the presence in the peroxidase system
HCO3creates the strongest buffer in the saliva buffer system: bicarbonate/carbonic acid
PO43-, HPO42-, H2PO4involved in the maturation of enamel remineralization of initial damage
form a buffer system phosphate/phosphoric acid
play an important role in the formation of tartar (calculus)
13
2017-01-12
The role of saliva:
water
antioxidant
function
lingual lipase, salivary amylase
peroxidases,
catalase,
uric acid
cystatin, histatins,
PRPs, statherin,
PO43-, HPO42-, H2PO4-,
F-, Ca2+
buffering
function
PRPs
mucin, histatin,
peroxidases,
immunoglobulins,
cystatins, lactoferrins,
lysozyme
PO43-, HPO42-, H2PO4,
HCO3-, urea
mucins, statherins
Moisturizing function of saliva:
- facilitates the formation and swallowing bites, chew food
- reduces the deleterious effects of mechanical, chemical, thermal
and biological injuries of mucous membranes.
Protective function of saliva
Salivary proteins which covers the thin shell (called pellicle) surfaces
of the teeth and mucous membranes.
The pellicle includes:
amino acids and proteins selectively adsorbed on the tooth surface by the
interaction between the saliva proteins and hydroxyapatite of tooth enamel.
lactoferrin, lysozyme, amylase.
Pellicle role:
prevents continuous deposition of salivary calcium phosphate
protects the tooth from the acids produced by oral microorganisms after
consuming carbohydrates
forms in seconds after a tooth is cleaned or after chewing.
14
2017-01-12
Buffering function of saliva
At pH 6.4 - 7.8 saliva is supersaturated solution of calcium phosphates
After a small demineralisation lost minerals may return from the saliva of the
hard tissues of the tooth
10 2+ + 6 43- + 2
10
4)6
2
(The balance between saliva and enamel.)
Acidification of the environment (eg. due to fermentation of sugars by
enzymes) increases the solubility of enamel (saliva reduces the degree of
saturation of calcium phosphate, saliva becomes unsaturated solution).
in the acidic medium: phosphate groups of hydroxyapatite may be associated
with a proton:
2+
10
4)6
2
9 2
4)6
2
(Connection of two H+ corresponds to the release of one Ca2+ ion grom crystal apatite - demineralization of enamel.)
Binding of Ca2+, PO43-, F- (toothpaste) induces an acid-resistant fluoroapatite in
place of the primary hydroxyapatite (remineralization of enamel).
Salivary buffers:
Buffer systems maintain the acid-base balance by neutralizing organic acids
(from food and produced by cariogenic bacteria).
The most important salivary buffers are:
bicarbonate buffer (higher concentration in stimulated saliva):
HCO3- + H3O+ H2CO3 + H2O
phosphate buffer (higher concentration of unstimulated saliva):
2HPO42- + H3O+
2PO4 + H2O
saliva buffer capacity - mainly responsible is bicarbonate buffer
insufficient buffering capacity of saliva towards acids development of
caries, dissolving of the hydroxyapatite
15
2017-01-12
Saliva as a diagnostic material:
advantages saliva as a diagnostic material: availability and non-invasive way of sampling
indicators of the degree of risk of dental caries: amount of secreted saliva, buffer
capacity, content of microorganisms
Saliva is used for determining the concentration of:
steroid hormones,
drugs (benzodiazepines, theophylline, cyclosporine, paracetamol, gentamicin, cisplatin)
in toxicology to determine the concentration of the metals (lithium, cadmium, gallium)
addictive substances (drugs and their metabolites, such as amphetamine, cocaine , methadone,
phencyclidine, marihuana, opiates) - in the presence of saliva a few minutes after use and is kept
for 24 hours
alcohol - is detected in saliva after 20 minutes of ingestion and maintained for 12 hours
diagnosis of certain diseases, including cancer, cardio-vascular, autoimmune, infectious
(HIV, measles, rubella, viral infection of the liver) and gastrointestinal diseases
forensic medicine
Gingival crevicular fluid:
Physiological transudate formed in the gingival
sulcus
Percentage composition plasma-alike
The main source of immune cells in the oral cavity
The biochemical analysis of the fluid - non
invasive means of assessing the response in oral
disease.
Mesered parameters:
bacterial enzymes, bacterial degradation products,
connective tissue degradation products, host mediated
enzymes, inflammatory mediators, extracellular matrix
proteins
higher levels of these parameters can occur in gingival
crevicular fluid in oral diseases
16
2017-01-12
Thank you for your attention.
17