Collagenases, elastase, cathepsins, dipeptidylpeptidases, and tryptase are proteolytic enzymes that can be biomarkers for periodontal diseases. Studies have found increased levels of these enzymes in gingival tissue, saliva, and gingival crevicular fluid of patients with periodontal diseases compared to healthy patients. Other potential biomarkers discussed include hydrolytic enzymes like beta-glucuronidase and cytosolic enzymes like aspartate aminotransferase. Markers of connective tissue degradation like hydroxyproline and glycosaminoglycans have also been studied but techniques for isolation and detection are too complex for clinical use. Overall, certain proteolytic enzymes show potential as

1. BIOMARKERS IN
PERIODONTAL
DISEASES

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

3. PROTEOLYTIC ENZYMES
Collagenases
Elastase
Cathepsins
Dipeptidylpeptidases
Tryptase

4.  Collagenases :
 Synthesis: Macrophages, neutrophils, fibroblasts
and keratinocytes
 Stimulation: Bacterial products and cytokines
(Birkedal-Hansen,1993; Reynolds 1996)
• PMN’S
• MACROPHAGESMMP-8
• FIBROBLASTS
MMP-1
TISSUE INHIBITORS
OF METALLOPROTEINASES
(TIMP)

5.  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)

6.  MMP-8
--untreated chronic periodontitis active
--healthy/treated chronic periodontitis inactive/latent
(Hayakawa et al 1994; Uitto et al 1990)

7.  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)

8.  LJP---MMP-1 predominates
Comparison with untreated or treated chronic
periodontitis or healthy patients
Significantly LESS ( Ingman et al 1993)
TIMP-1 ---INCREASED
No longitudinal study of salivary MMP’s

9.  GCF
Collagenases--
Gingivitis- correlates
(Kowashi et al 1979.,Overall and Sodek, 1987)
Periodontitis- correlates
(Golub et al 1976.,Hakkarainen et al 1988)

10.  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%

11.  ELISA
 Commercial diagnostic test kit
Periocheck

12. 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

13.  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)

14.  Saliva –no studies
--high levels of cystatins

15.  GCF
 Cathepsin B and L
 Eley and Cox 1996
 100% sensitivity & 99.83% specificity
 Good predictor of future attachment loss

16.  Aspartate proteinases
 Cathepsin D—correlation
(Ishikawa et al 1972)
 No longitudinal study

17.  Serine proteases
 Elastase
 Production—PMNs
 Cell—inactive form(bound with inhibitor)

18. Inhibition
α1- proteinase
inhibitor
(α1-PI)
α2- macroglobulin
(α2 – M)
Secretory
leukocyte protease
inhibitor
(SLPI)
Skin anti-
leucoproteinase
(SKALP)

19.  Degrade proteoglycans
 Activate latent collagenases
 Importance in periodontal pathology

20.  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)

21.  GCF
 Correlate (Eley and Cox 1993)
 Good predictor
Commercial diagnostic test kit
Prognostik

22. 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)

23. 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)

24.  Bacterial DDPs
 Cleave glyclyprolyl residues
 Collagen degradation
 Correlate (Eley and Cox 1995)
 Good predictors

25. 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)

26.  Sensitivity-92%
 Good predictor
 Potential diagnostic test kit

27. ALKALINE PHOSPHATASE
 PMN’s
 Bone –metabolism
Gilbert 2003—serum
Plagnit 2002—implants (GCF)
Bezerra 2010—saliva and GCF

28. OTHERS
 Acid-phosphatase
 Inflammatory cells
 GCF
 Does not correlate

29.  Myeloperoxidase(MPO)
Anti-bacterial enzyme—PMN’s
MPO activity---no correlation –disease severity
(Cao and Smith 1989)

30.  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

31.  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

32. ADVANTAGES
 Predictive value—cathepsin B,elastase,DDP’s,b-
glucuronidase
 Simple
 Read after short time
 Patient education

33. 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

34.  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

35. CYTOSOLIC ENZYMES
Aspartate 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 kit
Periogard

36. Lactate dehydrogenase(LDH)
 Correlation
 Lamster et al 1988

37. MARKERS OF CONNECTIVE TISSUE DEGRADATION
Component Breakdown product
Collagen Hydroxyproline
Collagen cross links
N-propeptide
GAGs Heparan sulphate
Chondroitin-4-sulphate
Chondroitin-6-sulphate

38.  Hydroxyproline –containing peptides
No human studies

39. 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)

40. METHOD OF ISOLATION AND DETECTION
 Biochemical techniques—difficult for chair-side
HPLC
GAGs—cellulose acetate extraction and staining
No longitudinal studies

41.  Problems
 Complex and expensive techniques
 Long collection time
 Normal cycle of synthesis and degradation of
connective tissue and bone
 Not suitable for chair side

42. CONCLUSION

43. 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

44. 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.

45.  Thank you