Biomarkers

BIOMARKERS IN PERIODONTAL DISEASES

CONTENTS Proteolytic enzymes Hydrolytic enzmes Cytosolic enzymes Markers of connective tissue degradation Conclusion References

PROTEOLYTIC ENZYMES

Collagenases

Elastase

CathepsinsDipeptidylpeptidases

Tryptase

Collagenases : Synthesis: Macrophages, neutrophils,

fibroblasts and keratinocytes Stimulation: Bacterial products and cytokines

(Birkedal-Hansen,1993; Reynolds 1996)

MMP-8 • PMN’S• MACROPHAGES

MMP-1 • FIBROBLASTS

TISSUE INHIBITORS OF METALLOPROTEINASES(TIMP)

Collagenase-2 (MMP-8), Collagenase-1(MMP-1), and Collagenase-3 (MMP-13) activity

Gingival tissue, saliva and GCF (Ingman et al 1994, Westerlund et al 1996)

Assay Method Studies

Collagen substrates

Biochemically Sorsa et al 1990

Mono-clonal antibodies

Immuno-mediated(ELISA*, Western Blot#)

*Ingman et al 1996;Matsuki etal 1996

#Killi et al 2002

Polyclonal antibodies

Immuno-mediated(ELISA, Western Blot)

MMP-8--untreated chronic periodontitis active--healthy/treated chronic periodontitis

inactive/latent

(Hayakawa et al 1994; Uitto et al 1990)

Saliva

Salivary levels of MMP-8, MMP-9

(Hayakawa et al 1994; Ingman et al 1996; Makela et al 1994; Matsuki et al 1996)

MMP-8 and disease severity (Sorsa et al 1994) Treatment and MMP-8,2 and 9 (Hayakawa et al 1994; Makela et al 1994)

TIMP-1—untreated v/s healthy controls (Hayakawa et al 1994; Matsuki et al 1996)

LJP---MMP-1 predominatesComparison with untreated or treated chronic

periodontitis or healthy patientsSignificantly LESS ( Ingman et al 1993) TIMP-1 ---INCREASED

No longitudinal study of salivary MMP’s

GCF

Collagenases--

Gingivitis- correlates(Kowashi et al 1979.,Overall and Sodek, 1987)

Periodontitis- correlates

(Golub et al 1976.,Hakkarainen et al 1988)

Active enzymes and enzyme inhibitor levels ( Larivee et

al.,1986)MMP-8

MMP-8 and treatment (Chen et al 2000)

Mantyla et al.,2002—chairside test system

Threshold of 1mg/L—sensitivity –93% & specificity of 91%

ELISA

Commercial diagnostic test kit Periocheck

CYSTEINE PROTEINASES Cathepsin B,L,H—family of intracellular cysteine

proteinases

Production – fibroblasts, macrophages (Kennett et al 1994) and osteoclasts (Vaes 1988)

Activity ---acidic pH

Intracellular degradation

Extracellular degradation (Dickinson 2002)

Particularly active during bone resorption

Cathepsin B localised within lysosomes and associated with the surface membrane of macrophages

Presence on collagen fibrils---- collagen degradation

Inhibition—1)α2- macroglobulin

2)tissue inhibitors—cystatins(Eley &Cox 1991)

Saliva –no studies

--high levels of cystatins

GCF Cathepsin B and L

Eley and Cox 1996

100% sensitivity & 99.83% specificity

Good predictor of future attachment loss

Aspartate proteinases

Cathepsin D—correlation (Ishikawa et al 1972)

No longitudinal study

Serine proteases

Elastase

Production—PMNs

Cell—inactive form(bound with inhibitor)

Inhibition

α1- proteinase inhibitor(α1-PI)

α2- macroglobuli

n (α2 – M)

Secretory leukocyte protease inhibitor

(SLPI)

Skin anti-leucoproteina

se (SKALP)

Degrade proteoglycans

Activate latent collagenases

Importance in periodontal pathology

Saliva

Low –periodontally healthy patients

--zero in edentulous patients(Pederson et al 1995)

o Not a good indicator for gingivitis---45% detectable

(Uitto et al 1996)

GCF

Correlate (Eley and Cox 1993)

Good predictor

Commercial diagnostic test kit

Prognostik

TRYPTASE

Large amount in gingival tissue Localized to mast cells—stabalized as an active

tetramer by association with heparin Released on cell degradation Cleave compliment components and activate latent

collagenase Correlates (Cox and Eley 1992)

DIPEPTIDYLPEPTIDASES (DPPS) DPP II –Acidic pH DPP IV– Alkaline pH

DPP II—lysosomal enzyme—fibroblast(gingival tissue)

--Macrophages (GCF smears) DPP IV – lysosomal enzyme— macrophages, t-lymphocytes, fibroblasts (Kennett et al

1994)

Gingival tissue and GCF

Bacterial DDPs

Cleave glyclyprolyl residues

Collagen degradation

Correlate (Eley and Cox 1995)

Good predictors

HYDROLYTIC ENZYMES

ß- Glucuronidase Lysosomal –PMN’s Acid hydrolase—marker for primary granule release

4-week period of experimental gingivitis --Result +ve--spirochaetes,P.gingivalis,P.intermedia -ve--cocci (Lamster 1992)

Sensitivity-92% Good predictor Potential diagnostic test kit

ALKALINE PHOSPHATASE PMN’s Bone –metabolismGilbert 2003—serum Plagnit 2002—implants (GCF)Bezerra 2010—saliva and GCF

OTHERS Acid-phosphatase

Inflammatory cells GCF Does not correlate

Myeloperoxidase(MPO)

Anti-bacterial enzyme—PMN’s

MPO activity---no correlation –disease severity (Cao and Smith

1989)

Lysozyme (muraminidase) —antibacterial enzyme

Body secretions---tears and saliva , GCF

Salivary---LOWER—chronic periodontitis & IDDM (Markkanen et al

1986) Do not vary between untreated LAP and healthy

controls

GCF

Untreated LAP patients higher than in healthy controls—treatment--reduce to normal

(Suomalainen et al 1996)

Decreased in chronic periodontitis and increased in LAP

Lactoferrin

None have diagnostic potential

ADVANTAGES Predictive value—cathepsin

B,elastase,DDP’s,b-glucuronidase Simple Read after short time Patient education

DISADVANTAGES Choice ---difficult—inadequate knowledge Determination of site & sampling period Moiety associated with inflammation--mask

association with destructive disease No account of biological control mechanism Cost

Choice of appropriate marker—cathepsin B,elastase,DDP’s,b-glucuronidase

—periodontal disease progression(longitudinal studies)

--Collagenase ,tryptase,ALP,MPO-- disease severity & activity

--Acid phosphatase,lysozyme,lactoferrin--none

CYTOSOLIC ENZYMESAspartate aminotransferase (AST)

Serum and CSF---tissue necrosis and cell death

Gingivitis (Persson et al 1990)

Disease severity (Imrey et al 1991)

Longitudinal studies (Chambers 1991)

Commercial test kitPeriogard

Lactate dehydrogenase(LDH)

Correlation

Lamster et al 1988

MARKERS OF CONNECTIVE TISSUE DEGRADATION

Component Breakdown product

Collagen HydroxyprolineCollagen cross linksN-propeptide

GAGs Heparan sulphateChondroitin-4-sulphateChondroitin-6-sulphate

Hydroxyproline –containing peptides

No human studies

Glycosaminoglycans (GAGs)

Cellulose –acetate electrophoresis

Non-sulphated GAG –hyaluronic acid—gingivitis

Sulphated GAG —chondroitin-4-sulphate—periodontitis

Cross-sectional studies

(Embery et al 1982;Last et al 1985)

METHOD OF ISOLATION AND DETECTION Biochemical techniques—difficult for chair-

side

HPLCGAGs—cellulose acetate extraction and

staining

No longitudinal studies

Problems Complex and expensive techniques Long collection time Normal cycle of synthesis and degradation

of connective tissue and bone Not suitable for chair side

CONCLUSION

Clinical uses of a predictive diagnostic test

To : Prevent destructive disease and progression Identify high-risk patients Target treatment to specific sites Monitor the effects of periodontal treatment

REFERENCES:

1. Diagnostic tests of periodontal disease activity. Periodontics 5th edition. Eley & Manson.

2. Diagnostic biomarkers of oral & periodontal disease. DCNA 2005 July ; 49(3): 551

3. Critical analysis of biomarkers in the current periodontal practice. JISP. Vol.15, Issue2, Apr-Jun 2011.

4. Carranza’s clinical periodontology. 10th edition.

Thank you