Transcript Биохимия жидкостей полости рта

Biochemistry oral fluid
Author - Associate Professor,
Department of Biochemistry,
E.A. Ryskina
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.
The chemical composition of
saliva
saliva
Dry residue
Inorganic substances
chlorides
sulfates
bicarbonates
Organic substances
proteins
fluoro
phosphates
albumins
Non-protein
nitrogencontaining
compounds
globulins
Free amino
acids
bactericidal
enzymes
enzymes
lysozymes
Alpha-amylase
urea
ammonia
creatine
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:
- salivary glands (in figure);
- cells of the oral mucosa;
- gingival fluid (leukocytes);
- blood;
- microbial cells.
Their number depend on the
condition of the oral cavity
and the whole organism.
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).
Some protein and non-protein substances
included in saliva
NAME
CONCENTRATION
Total protein
mucins
lysozyme
1,5 – 3,0 g/l
2,5 –2,7 g/l
0,18 g/l
uric acid
urea
ammonia
0,03 –0,17 mol/l
1,4 –3,0 mol/l
2,6 mol/l
cholesterol
glucose
lactate
0,08 – 0,39 mmol/l
0,62 –1,56 mmol/l
20 – 40 mg/l
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.
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.
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
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.
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.
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.
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.
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 Nterminal of the statherines.
• They inhibit the growth of aerobic
and anaerobic bacteria
with histatines .
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.
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 Oglycosidic 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).
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.
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.
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.
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.
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 1,4glycosidic bonds between Nacetylglucosamine and Nacetylmuramic acid.
N-acetylmuramic acid is a
polysaccharide cell
walls of bacteria.
DNase and RNase
• DNase and RNase disrupt nucleic acid
bacteria and viruses and thus exhibit
antiviral and antimicrobial effect.
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.
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.
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.
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.
Structure IgAs
IgAs is in connection with the Sglycoprotein (secretory component),
which protects it from degradation by
enzymes.
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)
Inorganic substances in saliva mmol/l
(T.P. Vavilovа)
substance
saliva
blood plasma
Na +
6,6 - 24
130 - 150
K+
12 - 25
3,6 - 5,0
Cl -
11 - 20
97 - 108
Ca2+
0,75 – 3,0
2,1 – 2,8
Pi
2,2 – 6,5
1,0 – 1,6
Pi total
3,0 – 7,0
3,0 – 5,0
НСО3-
20 - 60
25
SCN-
0,5 – 1,2
0,1 – 0,2
Сu2+
0,3
0,1
I-
0,1
0,01
F-
0,001 – 0,15
0,15
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.
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.
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.
The structure
of the
micelles
• The core of the micelles is insoluble calcium
phosphate Ca3(PO4)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).
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.
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.
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.
• 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.
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.
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.
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.
Myeloperoxidase of leukocytes is involved
in the formation reaction of hypochlorite
(bactericidal effect)
• Leukocyte myeloperoxidase catalyzes the
reaction: H2O2 + Cl‾→ H2O + OCl‾
• Hypochlorite (OCl‾), that produced in the
reaction, has more potent bactericidal effect
than hydrogen peroxide in ten times.
Thank you for your
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