1. Evolution of Biochemical
Diagnosis
HEENA SHARMA

2. CONTENTS
• INTRODUCTION
• OBJECTIVES
• BIOCHEMICAL ANALYSIS

3. • Periodontitis, is an immune inflammatory response
which arises from the interaction between the
periodontopathogenic bacteria and host.
• The course of periodontal disease is marked by
discontinuous pattern of disease activity and inactivity
showing exacerbation and remission.
INTRODUCTION

4. • Traditional clinical measurements (probing pocket depth,
bleeding on probing, clinical attachment loss, plaque index,
radiographs) used for periodontal diagnosis are often of
limited usefulness in that they are indicators of previous
periodontal disease rather than present disease activity.
• The 1990s have seen the emergence of a multitude of
diagnostic tests based on physical, chemical, microbiological
and immunological methodologies.

5. • The philosophy behind the emergence of such tests is that:
The earlier the active disease is diagnosed, the less invasive
treatment is required.
The patient also has a reduced chair time, reduced expense
and a better the long term prognosis.
• Furthermore, with the recognition that risk groups and
unpredictable disease patterns exist, the benefits of objective
testing for initial diagnosis and for the long-term
maintenance of periodontal patients become clear.

6. OBJECTIVES OF AN IDEAL
DIAGNOSTIC TESTS
1. Quantitative.
2. Highly sensitive method capable of analyzing a single
periodontal site in health as well as disease.
3. Reproducible.
4. Highly specific.
5. Simple to perform.
6. A rapid, one or two stage procedure.

7. 7. Non-invasive.
8. Versatile in terms of sample handling, storage and
transport.
9. Amendable to chairside use.
10. Economical.
11. Dependent upon simple and robust instrumentation.

8. BIOCHEMICAL ANALYSIS
• Biochemical test kits used in periodontics analyze the
gingival crevicular fluid (GCF).
• However, molecular analysis of GCF elution was time
consuming and laboratory based, technically demanding
collection of sample leading to a small volume of the
fluid (1–5 𝜇L).

9. • Despite these apparent diagnostic and technical
disadvantages, GCF was still considered as a candidate
potential oral fluid for the development of adjunctive
non invasive chair-side point-of-care diagnostic
technology.

10. • The markers of GCF can be divided into 3 groups:
 Host derived enzymes and their inhibitors.
 Inflammatory mediators & host response
modifiers
 Tissue breakdown products

11. Host Derived Factors/ Enzymes
Intracellular
PROTEOLYTIC
ENZYMES
HYDROLYTIC
ENZYMES
Collagenase
Elastase
Cathepsin-B
Cathepsin-G
Cathepsin-D
Dipeptidylpeptidases
Tryptase
Aryl sulphatase
B-glucornidase
Alkaline Phosphatase
Acid Phosphatase
Myloperoxidase
Lysozyme
Lactoferrin
Extracellular
MMPs
11

12. Alkaline Phosphate (ALP)
• ALP is a host derived enzyme, involved in maintenance
of alveolar bone and renewal of the periodontal ligament.
• Main source of ALP in GCF is neutrophils.
• Ishikawa and Cimasoni (1970) demonstrated that ALP
level in GCF is 3 times in site with periodontal pockets
as compared to serum.
• Nakashima (1996) found total amount of ALP level in
GCF was significantly higher in active sites.

13. β-glucoronidase (βG)
• Is a lysosomal enzyme found in the primary azurophilic
granules of neutrophils.
• It is involved in the destruction of noncollagenous
components of extracellular matrix and is considered as an
indicator of periodontal disease activity.
• Cross-sectional data clearly show elevated βG activity in
sites with more severe periodontal disease.

14. Cathepsin B
• In GCF, macrophages are the main producers of cathepsin
B.
• It has a role in intracellular degradation and turnover of
proteins in lysosomal system.
• Its GCF concentration were found to be elevated in patients
with periodontal disease, but lower in patients with
gingivitis.

15. Aspartate aminotransferace (AST)
• It is released into the extracellular environment after cell
death.
• GCF AST high levels is related to confirmed attachment loss
and are strongly associated with disease active sites than
inactive sites.
• Periogard is the chair side diagnostic system that detects
levels of AST in GCF.

16. Periogard
• This commercial test consists of a tray with two test wells for
each tooth, and appropriate reagent for conducting the test.

17. Principle

18. DISADVANTAGES
• In practice, the PerioGard assay suffers from poor
differentiation between colors.
• Is a relatively complex procedure involving multiple
steps.

19. POCKET WATCH
• It is one of the simplest tests for analysing Aspartate
amino transferase (AST) at the chair side.

20. • The tray is incubated for 10 minutes at room
temperature for colour development.
• Pocket watch provides an index of cell death.
• The rate of conversion of malachite green is directly
proportional to the AST concentration.

21. Principle
• The GCF sample paper strip is placed in a well on the
reagent coated test tray.
• In the presence of pyridoxal phosphate, AST catalyzes the
transfer of an amino group from cysteinesulfinic acid, by a
aketoglutaric acid to yield β-sulfinyl pyruvate and glutamate.
• β-sulfinyl pyruvate rapidly decomposes and releases
inorganic sulfite which react with malachite green to convert
from a green dye to colourless form.

22. Disadvantages
• It does not predict disease activity.
• False positive results may be obtained if any other
disease is associated with inflammation masking
periodontitis.

23. Elastase
• It is a potent proteolytic enzyme found in lysosomal
granules.
• Elastase levels in GCF increase with induction of
experimental gingivitis and decrease when plaque
removal is reinstituted.
• Active disease sites produce an elevated elastases level in the
GCF.

24. PROGNOSTIK
• Prognostik developed in 1993, measures the level of
MMPs such as elastases in the GCF.

25. Principle

26. Matrix Metalloproteinaases (MMP)
• They are the members of a large subfamily of zinc-
dependent and calcium-dependent proteolytic enzymes
involved in the remodeling and degradation of extraxellular
matrix components such as:
• Collagen
• Fibronectin
• Laminin
• Proteoglycan core protein

27. • MMP-8, MMP-9 are the main collagen degrading enzyme in
GCF and saliva.
• Studies have shown that levels of MMP-8, MMP-3, MMP-
13 are associated with periodontal disease progression.
• Longitudinal studies have shown a significant decrease in
GCF MMP-8 activity after successful teatment, indicates
that MMP-8 is a indicator of current disease status.

28. PERIOCHECK
• It is a rapid chairside test to detect the presence of neutral
proteases and is FDA approved.
• The presence of these enzymes has been implicated in
collagen breakdown which is an important feature of
periodontal disease.

29. Principle

30. • The quantity and intensity of the colour reaction is compared
to a standard colour chart and is related to the level of neutral
protease activity originally present in the crevicular fluid
sample.
• DRAWBACKS
 Interproximal sites cannot be sampled due to saliva
contamination,
 It is not specific for PMNL collagenase and may include
enzymes of bacterial origin.

31. MMP DIPSTICK TEST
• This forms the basis for the development of both qualitative
and quantitative chairside POC technologies.
• It help in the rapid detection of pathologically elevated
levels of MMP-8 in oral fluids and serum.

32. • Monoclonal antibodies for MMP-8 are being utilized in
chairside POC immunotests for oral fluid and serum MMP-8
analysis.
• The MMP-8 stick-test can differentiate healthy gingiva and
gingivitis sites from periodontitis sites.
• the results obtained correlates with that of quantitative
laboratory Immunofluorometric Assay (IFMA)

33. Immune & Inflammatory Mediators
Immune response –
Antibody
Total IgG and IgG subgroup
Complement components
Inflammatory response –
Arachidonic acid derivatives – PGE2
Cytokines – IL-1, IL-2, IL-4, IL-6, TNF- α
33

34. Antibodies
• During the periodontal inflammation, antibodies are formed
against periodontopathogenic bacteria, have been identified
in the GCF of periodontitis patients.
• In advanced stages of periodontitis, IgG is the primary
immunoglobulin in GCF. Which indicates the IgG positive
plasma cell predominance in the lesion.
• IgA has been demonstrated to be a potent inhibitor of
complement activation by IgG.

35. • The GCF levels of IgA have been found to be elevated in
patients with severe periodontal disease.
• Studies have demonstrated that GCF level of IgM
significantly reduces following the periodontal treatment.

36. Cytokines
Saliva: Potential cytokine marker in saliva is PAF(platelet
activation factor).Salivary PAF is significantly higher in
untreated chronic periodontitis pts, correlate with clinical
indices of disease severity & also reduces following treatment.
(Rasch et al,Garito et al 1995)
GCF: IL-1β & TNF- α in GCF causes stimulation of
prostaglandin E2 & collagenase production -thus most
important in the pathogenesis of periodontitis
36

37. IL-1 &  are present in inflamed gingiva and the
concentrations increase with severity and show decrease
in their levels following treatment.
Levels of IL-6 and IL1 high in patients with refractory
periodontitis.
TNF in GCF does not correlate with probing depth/
gingival inflammation (Rossomando et al)
37

38. Lee et al( longitudinal study): Levels of IL-1beta, IL-
2,4,6 and TNF-α might associate with but not predict the
progressive attachment loss.
But in the same study in areas of bone loss, investigators
found increased levels of IL-2,6 and IL-1beta – suggested
that these markers may be predictive of future attachment
loss in refractory periodontitis.

39. Since monoclonal antibodies have been produced for
these cytokines, they can be measured by ELISA- thus
potential for use in diagnosis.
The level of predictive ability of these cytokines is
unclear.
39

40. Prostaglandins
Prostaglandin E2 stimulates fibroblasts and osteoclasts to increase
the production of MMPs. IL-1β and other cytokines.
GCF PGE2 levels are low in health.
Increase in experimental gingivitis [between 32 & 53 ng/ml] &
significantly higher in untreated periodontitis
GCF PGE2 is predictive for periodontal disease activity.
40

41. TOPAS Tm (toxicity prescreening assay)
• It is used to detect elevated levels of bacterial toxins and
increased levels of human and bacterial inflammatory
proteins.
• The first generation TOPAS was a manual test and the
latest second generation TOPAS TM is an automated one.
• It is a simple, painless test, performed within 7 minutes.

42. Principle
TOPAS is based on the reaction of bacterial toxins in GCF with
a specially developed mixture of chemical reagents to produce a
dose related color change.
The greater the concentration of toxins in the GCF sample, the
brighter the yellow color of the assay mixture.
42

43. Tissue Break Down Products
Fibronectin: Cleaved components of fibronectin
Collagen: Hydroxy proline, collagen cross linkage peptides,
terminal peptides
Proteoglycan: GAGs
GAGs: Heparin sulphate, chondroitin-4-sulphate, chrondroin 6-
sluphate.
43

44. Fibronectin
• Fibronectin is a major component of extracellular matrix,
produced by degradation of ECM.
• Researches have revealed that fibronectin is invariably found
in degraded for in GCF.
• However, its potential use as a biomarker is still to be
established.

45. Hydroxyproline
• It is a major product of collagen breakdown.
• Akalin et al (1993) investigated the hydroxyproline and total
protein level in gingiva and GCF of patients with juvenile,
rapidly progressive and adult periodontitis and found the
level of both proteins were raised.

46. Glycosaminoglycans (GAGs)
• The levels of GAGs in GCF are good indicators of
underlying tissue turnover.
• Studies were done and found the increased level of GAGs in
GCF during inflammation and periodontal destruction.
• However, more research is required to established GAGs as
reliable indicator of periodontal disease progression.

47. MARKERS OF BONE RESORPTION
• The components of bone that could be released during
bone resorption & are present in GCF are known as bone
specific proteins.
They are
1. Osteonectin
2. Bone phosphoprotein (N-propeptide)
3. Osteocalcin
4. Telopeptides of type1 collagen.
47

48. osteonectin
• it is the most abundant glycoprotein secreted by human
osteoblas in developing bone and odontoblasts in
developing teeth.
• Its level in GCF as biomarker for periodontal disease
progression have been investigated. However, more
evidence is required to establish its use as a marker for
periodontal breakdown.

49. osteocalcin
• It is a calcium binding protein of bone, synthesized by
osteoblasts.
• It play a important role in bone resorption and
mineralization.
• It is an important biomarker of bone turnover.
• Cross-sectional study has shown an association between
the GCF osteocalcin levels and periodontal disease.

50. Osteopontin
• It is a phosphorylated, sialic acid rich, non-collagenous
bone matrix protein.
• It is produced by both osteoblasts and osteoclasts.
• Its level is increased in active sites of bone metabolism.
• Studies have demonstrated that OPN level in GCF
increase with the progression of periodontal disease.

51. Pyrolidine cross-links( ICTP)
• It is derived from the carboxy terminal telopeptide or
deoxypyridinoline.
• It has emerged as an promising biomarker of bone
resorption in an array of osteolytic diseases.
• Many investigations have demonstrated the relationship
between ICTP and high/low bone turnover disease such
as post-menopausal osteoporosis.

52. • The GCF levels of ICTP have been associated with bone
and attachment loss in periodontitis.
• Reaearch has also shown elevated levels of ICTP in
periodontitis cases exhibiting high levels of putative
periodontal pathogens including, T forsythia, P gingivalis,
F nucleatum sub species.

53. Sampling & detection
Osteonectin in GCF – Nitrocellulose strip
 Osteocalcin - conventional paper strip for 30 secs. or multiple
collection for 1min at same site
Diagnostic tests:
Detection by specific monoclonal or polyclonal antibodies -
ELISA
Osteocalcin - ELISA (Nakashima1994) & radioimmunoassay
(Giannobile., 1995)
Osteonectin & N-propeptide – ELISA (Bowers.et.al 1989)
53

54. • In 21st century saliva become the medium of choice because the collection of
saliva samples was considered to be:
• more convenient
• Practical
• rapid
• non invasive
• requires neither professional stuff nor specific materials.
• Saliva and mouth-rinse represented a pooled sample from all periodontal sites
providing an overall assessment of periodontal disease and health at subject
level.

55. SALIVARY BIOMARKERS
• Secretions from the major salivary glands (parotid,
submandibular and sublingual), which have a large number
of proteins and peptides, are responsible for maintaining the
integrity of the oral cavity.

56. Marker Relationship with periodontal
disease
Type of periodontal disease
SPECIFIC
Immunoglobulins (IgG, IgA,
IgM)
Interfares with adherence and
bacterial metabolism /
increased concentration in
saliva of periodontal patients
Chronic and aggressive
periodontitis
NONSPECIFIC
Mucins Interfares with colonization of
A.a
Aggressive
Lysozyme Regulation of biofilm
accumulation
chronic
Lactoferrin Inhibit microbial growth/
increase co-relation with A.a
Aggressive
Histatin Neutralize liposaccharide and
enzyme known to effect the
periodontium
Chronic and aggressive
Peroxidase Interfares with biofilm
accuulation
chronic

57. PERIO 2000

58. • The Diamond Probe/Perio 2000 System is a
periodontal probe that combines advanced ion
selective electrode technology with the standard
"Michigan O" style probe.
• It is intended to measure: probing depths
 to evaluate the presence or absence of bleeding or
probing.
detect the presence of sulfides in periodontal pockets.

59. • The sulfide sensor component in the system is used as an
adjunct to traditional diagnostic techniques in the
evaluation of periodontal diseases in adult patients.
• The system consists of:
Single use, disposable sensor tips that combine an
updated standard Michigan "O" style dental probe with a
sulfide sensor for use during one-time examinations .
An electronic control unit that provides real-time visual
feedback of bacterial activity to the practitioner and
patient .

60.  Probe handle, hand-piece cable, foot switch, and
external power supply
Wash solution
Accessory stand for convenient wash cup placement and
temporary probe storage
 System check

61. Principle
Control unit reports the sulfide level at each site
In a digital score from 0.0-5.0
Sulfide sensing element generates an electrochemical voltage
Proportional to the concentration of sulfide ions present
Diamond probe is placed directly into the periodontal pocket or
tongue

62. CONCLUSION
• In periodontology, the success of any treatment is dependent
upon the accuracy of the initial diagnosis.
• Many of the biochemical chairside diagnostic test kits have
been marketed. The newly commercially available chairside
tests for host and bacterial markers of periodontal disease
offer prospects which would make the monitoring of specific
sites possible.
• However, the clinician must ensure that the use of such tests
will benefit the patients in terms of both the value of
diagnostic data obtained and the cost in time and money.