Effects of Low-dose aspirin and gum diseases

Periodontal conditions in relation to
low-dose aspirin therapy
in ex- and non-smokers

by

Arthur Drouganis BDS, Grad Cert Dent

A thesis submitted for the degree of
Master of Dental Surgery
(Periodontics)

The University of Adelaide
Dental School
November 1999

Dedication

This thesis is dedicated to my loving wife Helen, and

my children Vicky, Lambros and Margaret whose

support, enthusiasm and tolerance enabled me to

complete the work.

TABLE OF CONTENTS
Acknowledgments ............................................................................................................xi

Glossary of terms...........................................................................................................xiii

Summary.........................................................................................................................xiv

Chapter 1 Introduction....................................................................................................1

Chapter 2 Review of the literature..................................................................................5

2.0 SUMMARY OF THE PRESENT UNDERSTANDING OF THE INFLAMMATORY RESPONSE..............................5
2.1 ENDOGENOUS MEDIATORS OF INFLAMMATION...............................................................................8
2.1.1 Histamine: ...................................................................................................................8
2.1.2 Bradykinin: ..................................................................................................................9
2.1.3 Plasmin: .....................................................................................................................9
2.1.4 Complement: ...........................................................................................................10
2.1.5 Platelets:....................................................................................................................11
2.2 EICOSANOIDS ........................................................................................................................12
2.2.1 General properties of eicosanoids.............................................................................13
2.3 ROLE OF EICOSANOIDS IN PERIODONTAL TISSUES.........................................................................15
2.3.1 Biosynthesis of eicosanoids.......................................................................................16
2.3.2 Arachidonic acid pathways: eicosanoid production....................................................17
2.3.3 Catabolism of the eicosanoids...................................................................................24
2.4 THE ROLE OF CYTOKINES IN PERIODONTAL TISSUES.....................................................................25
2.5 CELLULAR EVENTS IN INFLAMMATION........................................................................................31
2.5.1 Macrophage phenotypes...........................................................................................32
2.5.2 Alveolar bone resorption ..........................................................................................33
2.6 NONSTEROIDAL ANTI-INFLAMMATORY DRUGS IN PERIODONTAL DISEASES.......................................34
2.6.1 History of salicylates..................................................................................................34
2.6.2 Physio-chemical properties of aspirin and other salicylates ......................................35
2.6.3 Periodontal studies of the effects of NSAIDs over the last 20 years..........................39
2.7 PERIODONTAL STUDIES WITH ASPIRIN.........................................................................................52
2.7.1 The Waite study: .......................................................................................................52
2.7.2 The Feldman study ...................................................................................................53
2.7.3 The Flemmig study ...................................................................................................54
2.7.4 The Heasman study .................................................................................................55
2.8 SMOKING AND PERIODONTAL DISEASES.......................................................................................56
2.8.1 The periodontal effects of past smoking and smoking dose......................................57

i

2.9 PERIODONTAL MEASURES..........................................................................................................58
2.9.1 The experimental unit................................................................................................59
2.9.2 Measurement of extent and severity of periodontal attachment loss.........................59
2.10 NULL HYPOTHESES................................................................................................................61

Chapter 3 Materials and methods.................................................................................62

3.1. SAMPLE SELECTION.................................................................................................................62
3.2 QUESTIONNAIRE......................................................................................................................64
3.3 ORAL EXAMINATION...............................................................................................................66
3.4 CLINICAL MEASUREMENTS........................................................................................................66
3.4.1 Plaque Index .............................................................................................................66
3.4.2 Calculus ....................................................................................................................67
3.4.3 Bleeding index. ........................................................................................................67
3.4.3 Tooth mobility ...........................................................................................................68
3.4.4 Furcation involvement ...............................................................................................69
3.5 DETAILS OF THE STUDY............................................................................................................69
3.5.1 Periodontal attachment loss (PAL).............................................................................69
3.5.2 Periodontal Pocket Depths (PPD)..............................................................................70
3.5.3 Gingival Recession (GR)...........................................................................................70
3.5.4 Examiner standardisation:.........................................................................................70
3.5.5 Procedure..................................................................................................................70
3.6 STATISTICAL METHODOLOGY....................................................................................................71

Chapter 4 Results............................................................................................................72

4.1 INTRA-EXAMINER ERROR..........................................................................................................72
4.2 PROFILE OF STUDY POPULATION:...............................................................................................72
4.3 DEMOGRAPHICS......................................................................................................................73
4.3.1 Age categories of subjects.........................................................................................73
4.3.2 Education status of the subjects................................................................................74
4.3.2 Oral health behaviour................................................................................................75
4.4 TOOTH LOSS...........................................................................................................................76
4.5 THE PERIODONTAL STATUS OF THE STUDY POPULATION................................................................76
4.6 ASSOCIATIONS OF ASPIRIN AND EX-SMOKING WITH VARIOUS MEASURES OF PAL............................78
4.6.1 The associations of aspirin and ex-smoking with mean PAL.....................................78
4.6.2 The associations of aspirin and ex-smoking on the extent and severity of PAL ........79
4.6.3 Associations of aspirin and ex-smoking with the most severe site of PAL (MSS-PAL)
.............................................................................................................................................81
4.6.4 Associations of aspirin and ex-smoking with the extreme worst site of PAL (EWS-
PAL).....................................................................................................................................82

ii

4.7 THE ASSOCIATIONS OF VARIOUS CLINICAL PARAMETERS ON MEAN PAL........................................84
4.7.1 Site and tooth variations in recession and pocket depth by mean PAL......................85
4.7.2 Socio-economic factors and periodontal attachment.................................................86

Chapter 5 Discussion....................................................................................................115

5.1 PROFILE OF THE STUDY POPULATION.......................................................................................115
5.1.1 Age groupings .........................................................................................................117
5.2 QUESTIONNAIRE....................................................................................................................119
5.2.1 Socio-economic status.............................................................................................120
5.3 PERIODONTAL ATTACHMENT LOSS..........................................................................................121
5.3.1 Age associations with PAL .....................................................................................121
5.4 MEASURING PAL.................................................................................................................122
5.4.1 Case definitions.......................................................................................................122
5.5 OUTCOMES OF ASPIRIN AND PAST SMOKING ON PAL................................................................125
5.5.1 Mean PAL................................................................................................................125
5.5.2 MSS-PAL.................................................................................................................127
5.5.3 EWS-PAL.................................................................................................................127
5.5.4 Plaque .....................................................................................................................128
5.5.5 Gingival bleeding ....................................................................................................129
5.6 COMPARISONS WITH OTHER ASPIRIN STUDIES............................................................................130
5.7 SMOKING AND PAL..............................................................................................................133
5.8 PREVALENCE OF PERIODONTAL ATTACHMENT LOSS...................................................................133
5.9 FUTURE RECOMMENDATIONS...................................................................................................134

Conclusions....................................................................................................................135

Appendix A....................................................................................................................137

Appendix B.....................................................................................................................138

Appendix C....................................................................................................................139

Appendix D....................................................................................................................140

References......................................................................................................................144

iii

TABLES

Table 2.1 Interactions of plaque bacteria and their products in inflammation and

immunity............................................................................................................................7

Table 2.2 Composition of eicosanoids ...........................................................................12

Prostanoids.......................................................................................................................12

Table 2.3 Cell sources and actions of prostanoids .......................................................15

Table 2.4 Major tissue destructive mediators in periodontitis ...................................26

Table 2.5 Neutrophil components and function (Williams et al. 1996)......................31

Table 2.6 The types of NSAIDs (and their classification) used in periodontal studies

...........................................................................................................................................39

Table 2.7 Periodontal effects of NSAIDs in human studies.........................................40

Table 3.1 Inclusion and exclusion criteria.....................................................................63

Table 3.2 Aims of questionnaire. ...................................................................................65

Table 3.3 Plaque index ...................................................................................................67

Table3.4 Modified Sulcus Bleeding Index (mSBI).......................................................67

Table 3.5 Tooth mobility index......................................................................................68

Table 3.6 Furcation index...............................................................................................69

Table 4.1 Intra examiner reliability test using kappa statistics..................................87

iv

Table 4.2 The number and percentage distribution of subjects participating in the

study by group.................................................................................................................87

Table 4.3 Distribution of age by group..........................................................................87

Table 4.4 A Scheffés analysis of homogeneity between two groups at a time for

mean age differences.......................................................................................................88

Table 4.5 Scheffésa, analysis for homogeneity between subsets..................................88

Table 4.6 Demographics on pension status with group specific characteristics........89

Table 4.7 Pension status in relation to denture use. ....................................................89

Table 4.8 Demographic data on schooling of all subjects with group specific

characteristics. ................................................................................................................89

Table 4.9 A self-evaluation of English language skill...................................................90

Table 4.10 Socio-economic factors and dental behaviours..........................................90

Table 4.11 Population and percentage distribution of subjects since their last dental

visit. The time range was from less than one year to never visiting the dentist.......91

Table 4.12 Missing teeth by age and group...................................................................92

Table 4.13 Missing teeth and smoking history..............................................................92

Table 4.14 The mean plaque index per group. ............................................................92

Table 4.15a Profile of aspirin use and subject numbers .............................................93

Table 4.15b The association of age and past smoking on mean plaque scores with

tests of significance..........................................................................................................93

v

Table 4.16 Distribution of mean percentage of teeth with calculus by age, aspirin

and ex-smoking. ..............................................................................................................94

Table 4.17 The association of age with mean percentage of calculus between groups

...........................................................................................................................................95

Table 4.18 The association of low-dose aspirin and ex-smoking with the mean

percentages of mobile teeth............................................................................................95

Table 4.19 The correlation of low-dose aspirin and past smoking with mean PAL..96

Table 4.20 The association of aspirin dosage with mean PAL....................................97

Table 4.21 The association of aspirin duration with mean PAL.................................97

Table 4.22a The association of past smoking dosage and duration with mean PAL.

...........................................................................................................................................97

Table 4.22b The correlation of the number of cigarettes smoked and duration of

smoking with mean PAL with t-test of significance.....................................................98

Table 4.23 Univariate analysis of variance in mean PAL at ≥2, ≥4 ≥ 5 and ≥7mm.

...........................................................................................................................................99

Table 4.24 Univariate analysis of variance on mean % PAL at ≥2, ≥4, ≥ 5 & ≥7mm.

.........................................................................................................................................100

Table 4.25 The magnitude of the association of aspirin and smoking history with

severity and extent of PAL at ≥2, ≥4, ≥ 5 & ≥7 mm PAL using the general linear

model (2-way ANOVA) of analysis..............................................................................101

Table 4.26 The correlation of aspirin and past smoking history with MSS-PAL.. .102

vi

Table 4.27 The age class distribution of males 50+ years in metropolitan Adelaide in

1996 from census statistics and their appropriate frequency distribution. ............103

Table 4.28 The proportional weights given to each group using the percentage

frequency of each class interval from census statistics for metropolitan Adelaide.103

Table 4.29 Descriptive statistics of EWS-PAL ...........................................................103

Table 4.30 The association of aspirin and past smoking history with EWS-PAL

using weighted data.......................................................................................................104

Table 4.31 The ratio of aspirin to smoking on various measurements of PAL. ....105

Table 4.32 Associations of plaque and age with mean PAL with tests of significance.

.........................................................................................................................................105

Table 4.33 Associations of calculus and age with mean PAL with tests of

significance.....................................................................................................................105

Table 4.34 Associations of gingival bleeding and age with mean PAL with tests of

significance.....................................................................................................................106

Table 4.35 Socio-economic factors, oral hygiene patterns and mean PAL (mm)....106

Table 4.36 The statistical power values for most ANOVA analyses ........................107

Table 4.37 Relative percentage of subjects with medical conditions per group......107

Table 4.38 Outcome of age, ex-smoking and aspirin with various indices of PAL. 107

vii

Figures

Leukotrienes.....................................................................................................................12

Figure 2.1 Products and pathways of cyclo-oxygenase ...............................................14

Figure 2.2 The chemical structures of PGE2 and TxB2 .............................................20

Figure 2.3 Structure of aspirin ......................................................................................36

Figure 2.4 Effects of aspirin on cyclo-oxygenases .......................................................38

Figure 3.1 A copy of an advertisement placed in local press media to recruit

subjects.............................................................................................................................62

Figure 4.1 The mean percentage of sites with gingival bleeding (modified bleeding

index)..............................................................................................................................108

Figure 4.2 The mean percentage of teeth with calculus ............................................108

Figure 4.3 Cumulative distribution of MSS-PAL representing the worst score (site)

per tooth per subject, averaged over all subjects. .....................................................109

Figure 4.4 Diagrammatic representation of PAL according to smoking and aspirin

taking history, showing mean PAL, MSS-PAL and EWS-PAL...............................110

Figure 4.5 Cumulative distribution of EWS-PAL. Data were weighted using age

class statistics for metropolitan Adelaide population................................................111

Figure 4.6 Variations of recession and pocket depths by tooth- and jaw type for the

whole study population.................................................................................................112

viii

Figure 4.7 Variation of recession and pocket depths by tooth- and jaw type in the

AXS group......................................................................................................................112


NAXS group. .................................................................................................................113


ANS group......................................................................................................................113


NANS group...................................................................................................................114

ix

Signed Statement

This research report is submitted in partial fulfillment of the requirements of the Degree

of Master of Dental Surgery (Periodontics) in the University of Adelaide.

The thesis contains no material which has been accepted for the award of any other

degree or diploma in any University and that, to the best of my knowledge and belief, the

thesis contains no other material previously published or written by another person,

except where due reference is made in the text of the thesis.

I give consent to this copy of my thesis, when deposited in the University Library, being

made available for photocopying and loan if accepted for the award of the degree.

……………………………………..

Arthur Drouganis.

November 1999

x

Acknowledgments

I wish to take this opportunity to thank those people who have assisted me in completing

my candidature. I am particularly grateful to many people but utmost to my wife, and

family for their patience and understanding throughout this challenging course.

I am truly indebted to two individuals. Robert Hirsch my supervisor, a true researcher,

for his kindness, knowledge and in particular his insight and wisdom who lent me

unconditional support, tempered at times, by considerable forbearance. To Bryon

Kardachi, for his clinical knowledge, expert guidance and for his enthusiasm. The

knowledge I have gained from both of them is, and will be invaluable.

My thanks go to the Colgate Australian Clinical Dental Research Centre for the use of its

state-of-the-art facilities and I am especially grateful to Kerrie Ryan and Jane Burns who

gave excellent support and assistance. To Colgate Australia for their generosity in

supplying the Oral Care Kits which were given to each participant in the study. A

special thank you to Professor Felix Bochner, Department of Clinical and Experimental

Pharmacology, Division of Health Sciences University of Adelaide for his initial

guidance.

I am deeply grateful to Knute Carter for his meticulous statistical analyses of the data.

To Jane Carter for her enthusiasm and ideas on the study

These people have inspired and encouraged me to ask questions, to learn to reason and

think independently. I truly believe I have been educated.

Thank you.

xi

" Do not be rash to make friends; but, when once they are made, do not drop them"

DIOGENES (412-332 B.C.)

A Greek philosopher

I can quite honestly say that I have made life time friends.

xii

Glossary of terms

ANS Aspirin Never Smoked group
AXS Aspirin eX-Smoker group
COX Cyclo-oxygenase, an enzyme that produces the prostanoid and
thromboxane mediators of inflammation
Cytokines Polypeptide mediators released by cells involved in inflammation
healing and homeostasis
EWS-PAL The extreme worst site of PAL per subject, then averaged across
each group
Extent The proportion of tooth sites of an individual with PAL exceeding
1mm and often measured at various threshold values
GCF Gingival Crevicular Fluid
IgG Immunoglobulin-G
IL-1 Interleukin-1 an inflammatory cytokine involved in inflammation,
immunity, tissue breakdown and homeostasis
IL-6 Interleukin-6 an inflammatory cytokine involved in inflammation,
immunity, tissue breakdown and homeostasis
Low-dose aspirin ≤300mg per day
LPS Lipopolysaccharide
Mean PAL The average PAL of all sites per subject, then averaged across
each group
MSS-PAL The most severe site of PAL per tooth per person then averaged
across each group
NANS No Aspirin Never smoked group
NAXS No Aspirin eX-Smoker group
NSAIDs Non-steroidal anti-inflammatory drugs
PAL Periodontal attachment loss
PGE2 Prostaglandin-E2. A primary cyclo-oxygenase mediator of
inflammation
Prevalence The proportion of group who have PAL (ie cases)
Severity The degree of PAL averaged per affected tooth sites
TNF-α A proinflammatory cytokine with synergistic effects with other
cytokines

xiii

Summary

In the 1970's, Vane proposed that the anti-inflammatory effects of aspirin and aspirin-like

drugs (non-steroidal anti-inflammatory drugs, NSAIDs) were due to inhibition of the enzyme

cyclo-oxygenase, which stops the production of prostanoids (prostaglandins and

thromboxanes). By the early 1980's, high doses of aspirin and other NSAIDs were shown to

significantly reduce gingivitis, periodontal attachment loss and alveolar bone loss in humans.

However, long-term use of these agents in periodontal therapy was not advocated, due to their

side effects and the inconsistent findings between studies. Often test and control groups were

not from the same sample population, results were based on concurrent use of other NSAIDs,

dosages and duration varied between groups, and there was no control for smoking effects.

Research in the 1990's showed that periodontitis is a multifactorial disease, being dependent

on genetic and environmental influences, which modify the host response to the microbial

challenge. One of the primary environmental risk factors for periodontitis is cigarette

smoking. Ex-smokers lie between non-smokers and current smokers with regard to the

severity and extent of periodontal attachment loss and alveolar bone loss; people who quit

smoking respond to periodontal therapy similarly to non-smokers.

There is no information in the literature about the periodontal effects of low-dose aspirin on

the periodontium in either non-smokers or ex-smokers. The aim of this study was to assess the

periodontal status of a self-selected sample of men (aged 50 and above), residing in

metropolitan Adelaide, South Australia, with respect to aspirin intake and smoking history.

Subjects were targeted by advertisements placed in the local press.

Demographic data were collected from information obtained from a self-administered

questionnaire and periodontal health was assessed by a periodontal examination carried out by

one operator, blind to each subject’s aspirin and smoking history. Measurements of pocket

depths and gingival recession were made at six sites of all teeth present and were used to

xiv

compute periodontal attachment loss (PAL) for all subjects. Other parameters recorded were

plaque and calculus accumulation, gingival and bleeding indices and tooth mobility.

Periodontal assessments were carried out in 392 men, aged 50-85 years. Significant age

effects were found on PAL but these were of small magnitude in comparison to the significant

influences that aspirin and ex-smoking had on PAL. The subjects were divided amongst four

sub-groups:

• aspirin never smoked (ANS),
• aspirin ex-smokers (AXS).
• no aspirin never smoked (NANS)
• no aspirin ex-smokers (NAXS).

The extent and severity of PAL was evaluated against a background of age, ethnicity, socio-

economic and dentition status. The study population comprised low, middle and higher

educational levels and there were no significant distribution differences between the groups.

The study population comprised a much higher group of educated subjects when compared to

the general population of Adelaide. Higher educated subjects with good English skills

brushed more frequently and had a more recent scale and clean than the lower educated

groups. A measure of subjects’ economic level was their pension status; pensioners

representing low income. Approximately 58.9% of subjects were pensioners; there were no

significant differences in mean PAL between pensioners and non-pensioners.

In order to correlate the effects of aspirin and smoking habits on advanced PAL, three

measures of PAL were used; mean PAL, the most severe site of PAL (MSS-PAL) and the

extreme worst site of PAL (EWS-PAL). Mean PAL was the overall mean PAL of all sites

per tooth/per subject/per group. MSS-PAL was the most severe site of PAL of the six sites

per tooth/subject. This method associated the effects of aspirin and ex smoking on advanced

xv

PAL by reducing the overwhelming effects of sites with low PAL. EWS-PAL was the

extreme worst site of PAL/mouth. The results were as follows:

Mean PAL mm ± se MSS-PAL mm ± se EWS-PAL mm ± se
ANS 2.5 ± 0.01 3.7 ± 0.13 6.2 ± 0.22
AXS 2.8 ± 0.09 4.1 ± 0.11 7.0 ± 0.18
NANS 2.7 ± 0.08 4.0 ± 0.10 6.8 ± 0.17
NAXS 3.1 ± 0.08 4.4 ± 0.10 7.5 ± 0.17

Prevalence was measured using different threshold levels of PAL. Significant positive effects

of aspirin for the extent of PAL were found for all threshold levels. At thresholds of ≥2mm

PAL, the prevalence of PAL was approximately 94%. At a moderate threshold of 4mm PAL,

28.7% of subjects exhibited PAL ≥4mm with a mean severity score of 4.6 ± 0.03mm (se),

indicating that the percentage of subjects with advanced PAL was low particularly at higher

thresholds. Controlling for age, ANOVA analysis showed that the prevalence rate of PAL

was significantly lower in aspirin takers when compared to non-aspirin takers and these

effects were independent of smoking history. In addition, ex-smokers had significantly more

PAL compared to non-smokers and this effect was independent of aspirin history. The

prevalence of advanced PAL in subjects (using 7mm PAL as a threshold) was found to be

2.6% with a mean PAL of 7.7 ± 0.05mm (se).

Epidemiological studies (including this one) attribute all PAL to the effects of destructive

periodontal diseases. No account is given to other causes of PAL such as continuous tooth

eruption, alveolar dehiscence, cervical enamel projections, cracked or split roots and

retrograde periodontitis. Taking these factors into account, the true prevalence of advanced

PAL due to periodontitis within the community must be lower than the estimated rate of

10-15%.

My findings suggest that men aged 50 and above may benefit from taking low-doses of

aspirin daily in order to reduce their risk of PAL. With the reduced severity and extent

xvi

of PAL in ex-smokers taking aspirin, it is tempting to speculate that subjects with

periodontitis may benefit significantly by taking low-dose aspirin to reduce their

periodontal and cardiovascular risks, irrespective of their smoking history. Further

research should aim to establish whether patients with periodontitis would benefit from

taking low-dose aspirin as an adjunct to periodontal therapy and whether low-dose

aspirin modulates the effects of periodontitis in females and current smokers.

xvii

Chapter 1 Introduction

Destructive periodontal diseases are multifactorial in origin; the interplay between lifestyle

factors, the social environment and the dental biofilm determine an individual’s susceptibility

(Clarke and Hirsch 1995). Inflammatory as well as immunological responses are activated by

the many components of dental biofilm which constitutes the microbial challenge to the host

(Miyasaki 1996; Wilson and Kornman 1996; Darveau et al. 1997). The vascular and cellular

responses occurring in inflammation are controlled by the release of endogenous

inflammatory mediators (Page and Schroeder 1976; Page 1991; Genco et al. 1994;

Offenbacher 1996). There is an extensive list of endogenous inflammatory mediators known

to be involved in the regulation of the inflammatory response. In periodontal tissues, these

mediators are the link between health, tissue damage, inflammation and immunity (Page and

Schroeder 1976; Offenbacher et al. 1990; Page 1991; Offenbacher et al. 1993a; Offenbacher

et al. 1993b; Genco et al. 1994; Wilson and Kornman 1996).

One of the first and major pathways of tissue destruction in inflammatory periodontal

diseases is the synthesis and release of eicosanoids. Eicosanoids are formed from

membrane polyunsaturated fatty acids (mainly arachidonic acid), which include the

prostaglandins, prostacyclins, thromboxane A2 and the leukotrienes (Rang et al. 1996).

Eicosanoids are not found preformed in cells like histamine, but are generated de novo

from cell plasma membrane phospholipids when tissues are damaged (Salmon and Higgs

1987; Davies and MacIntyre 1992). They control many physiological and pathological

processes and are the most important mediators and modulators of the immuno-

inflammatory pathways (Rang et al. 1996). In response to microbial virulence factors,

damaged gingival tissues produce phospholipids, which become the substrate for

phospholipase. This enzyme synthesizes and releases free arachidonic acid (Howell and

1

Williams 1993) which may be synthesized into either prostanoids or leukotriene

products. These are associated with platelet aggregation, vasodilatation, chemotaxis of

neutrophils, increased vascular permeability and alveolar bone resorption. Prostanoids

are produced from arachidonic acid by cyclo-oxygenase (COX) which occurs in

neutrophils, macrophages, mast cells, fibroblast, lymphocytes keratinocytes, osteoblasts

and platelets (Howell and Williams 1993; Offenbacher 1996; Wiebe et al. 1996).

Leukotrienes are products produced by lipoxygenase and are restricted to neutrophils,

eosinophils, monocytes/macrophages and mast cells (Salmon and Higgs 1987).

The predominant prostanoid product in immuno-inflammatory responses in periodontal

diseases is prostaglandin E2 (PGE2) (Howell and Williams 1993; Offenbacher et al.

1993b). PGE2 is considered to be one of the key components in the pathogenesis of

periodontitis (Page 1991). A large portion of periodontal pathology is attributed to PGE 2,

especially in association with other proinflammatory cytokines (IL-1, IL-6, IL-8 and

TNF-α) (Alexander and Damoulis 1994; Mathur and Michalowicz 1997; Soskolne 1997;

Ellis 1998; Okada 1998). The principal sources of PGE2 in periodontal tissues are

macrophages, monocytes and fibroblasts (Fu et al. 1990).

In the 1970's, Vane (1971) advanced the hypothesis that the anti-inflammatory effects of

aspirin-like drugs lay in their ability to inhibit prostanoid synthesis (prostaglandins and

thromboxanes). Among its actions, aspirin irreversibly inhibits COX which exists in two

forms (Smith 1992; Meade et al. 1993; Vane 1994; Sharma and Sharma 1997; Dubois et

al. 1998):

• COX-1 is found in all cells as a constitutive enzyme, which produces the prostanoids

that regulate normal homeostasis (e.g. regulating vascular responses and coordinating

the actions of circulating hormones).

2

• COX-2 is the inflammatory cyclo-oxygenase that is induced only by inflammatory

stimuli, releasing prostaglandin E2 (PGE2). Platelets do not contain COX-2.

In the early 1980's, the effects of aspirin and other nonsteroidal anti-inflammatory drugs

(NSAIDs) on periodontal attachment loss started to be investigated in humans. People

taking high doses of aspirin or other NSAIDs were found to have significantly lower

plaque scores, less gingival inflammation, less attachment and bone loss than the controls

(Waite et al. 1981; Feldman et al. 1983; Williams et al. 1989; Jeffcoat et al. 1991;

Heasman et al. 1993b; Howell 1993; Offenbacher et al. 1993b; Flemmig et al. 1996;

Offenbacher 1996). NSAIDs were considered to have modified the host responses by

inhibiting PGE2 production and therefore reducing bone and periodontal attachment loss.

Unfortunately many factors in these studies were not controlled, such as age, sex, poor

comparison or control groups (sampling frame error), smoking and systemic disease.

Furthermore, most human studies were retrospective and often relied on the subjects'

recollection of dosage and duration, and more than one NSAID was often used

concurrently. The majority of aspirin studies used patients suffering from rheumatoid

arthritis who were taking high daily doses (650mg->3gm/day). These confounding

factors made comparisons between studies difficult and resulted in conflicting outcomes

with respect to plaque indices, gingival indices, periodontal attachment loss and alveolar

bone loss.

Low-dose aspirin's ability to irreversibly inhibit cyclo-oxygenase over the whole lifetime

of platelets has made it a widely used anti-thrombogenic agent in middle-aged and

elderly populations to prevent coronary artery disease, stroke and peripheral vascular

diseases, with low gastro-intestinal side effects (Vane and O'Grady 1993; Underwood

1994; Lloyd and Bochner 1996; Diener 1998; Müller 1998). Low-dose aspirin has

3

decreased the incidence of heart attacks and stroke by up to 50% (Vane 1994). Low-

dose aspirin can inhibit thromboxane A2 production by platelets equipotently as can

doses > 300mg. In Australia, the maximum benefit/risk ratio dose used is 100-150mg of

aspirin per day (Lloyd and Bochner 1996).

Smoking is recognised as the most important cause of preventable death and disease in the

western world (MacGregor 1992) and there is a clear association between smoking and the

prevalence and severity of PAL (Bergström and Floderus-Myrhed 1983; Haber et al. 1993;

Bergström and Preber 1994; Zambon et al. 1996). The greater the exposure in terms of pack

years, the greater the amount of PAL and alveolar bone loss (Grossi et al. 1996; Grossi et al.

1997).

To-date, no studies have investigated the effects of long-term low-dose aspirin on PAL.

Since there is a large pool of people in the community taking low-dose aspirin daily for

many years, this study was undertaken to correlate PAL with aspirin and smoking

histories. In particular, the aim of this study was to gather descriptive epidemiological

data relating to the extent and severity of periodontal attachment loss in an adult male

population within metropolitan Adelaide specifically targeting men with and without a

history of long-term low-dose aspirin therapy, with or without a history of smoking.

Data from this study could also provide information relating to oral hygiene habits,

dental attendance, socio-economic factors, tooth loss and attachment loss patterns in an

elderly population.

4

Chapter 2 Review of the literature

2.0 Summary of the present understanding of the inflammatory response.

Periodontal diseases are mostly chronic infections characterised by a destructive

inflammatory process affecting the supporting tissues of the tooth, with subsequent

pocket formation and resorption of the alveolar bone (Offenbacher 1996). The intent of

this review is to place the current understanding of the regulatory mechanisms that

influence the inflammatory response in perspective, focussing on prostaglandins as

important elements of the inflammatory process and as major mediators of periodontal

attachment loss (PAL) and alveolar bone loss (Offenbacher 1996; Gemmell et al. 1997;

Page et al. 1997).

Inflammation is the normal response of the body to infection, tissue injury or insult; it is

rapid and provides a first line of defence. It is initially a nonspecific host response,

eliciting the same reaction irrespective of the nature of the insult. The insult may be

microbial, physical or chemical in nature, and all initiate a series of local processes to

neutralise, limit the spread and eradicate the insulting agent(s) (Lakhani et al. 1993;

Offenbacher 1996; Gemmell et al. 1997; Page et al. 1997). Inflammation is divided into

acute and chronic forms based on the duration of the response and the predominant

inflammatory cell type. Whether acute or chronic, the process may be modified by many

environmental and host factors; such as the pathogenicity and virulence of the microbial

challenge, nutritional status, host immune status, use of antibiotics, anti-inflammatory

drugs and / or surgical/non-surgical therapy (Lakhani et al. 1993; Miyasaki 1996;

Wilson and Kornman 1996; Page et al. 1997). These responses are characterised by

dilatation of the local blood vessels, increased permeability of capillaries, plasma

exudate, with the chemotactic accumulation of neutrophils, monocytes/macrophages,

5

eosinophils, basophils and mast cells to the site of injury or infection (Kay 1970;

Bienenstock et al. 1986; Faccioli et al. 1991; Page et al. 1997). The chemotactic factors

are both chemotactic and cell activating, leading to increased cell numbers and / or

affinity of adherence receptors on the surfaces of both endothelial and inflammatory cells

(Page 1991; Page et al. 1997). The expression of adhesion receptors enables the

migration of inflammatory cells from the circulation into the sites of injury (Page 1991;

Page et al. 1997), where they actively eliminate the noxious agent and participate with

resident tissue cells in wound healing and tissue remodelling (Miyasaki 1996; Wilson

and Kornman 1996; Page et al. 1997). In addition to the cellular response, plasma

constituents including complement and immunoglobulins are poured into the sites of

inflammation (medications are also transported to these sites by the plasma or

inflammatory exudate).

The host through the neutrophils and macrophages has the capacity to destroy all

biological structures (Williams et al. 1996). In the process of containing the microbial

challenge, host defences can cause bystander tissue destruction which can be more

offensive than the original insult (Page et al. 1997; Okada 1998). The damage is either

essential, such as the removal of collagen allowing room in the tissue for an

inflammatory cell infiltrate, or the damage may be bystander damage (accidental) in the

process on containing the microbial challenge. "Bystander damage" is a common feature

of chronic inflammatory diseases such as rheumatoid arthritis, tuberculosis, and

emphysema. Loss of periodontal attachment in periodontitis is caused by bystander

damage from the host response to the microbial plaque (Williams et al. 1996; Page et al.

1997).

6

Inflammatory reactions consist of two components, the inflammatory exudate (the

plasma component) and the cellular response. Both responses are activated by the many

constituents of dental plaque biofilm which constitutes the microbial challenge to the

host in periodontal diseases (Miyasaki 1996; Wilson and Kornman 1996; Darveau et al.

1997). Aerobic and anaerobic bacteria found in the gingival crevice or periodontal

pockets release a variety of products that can cause the onset of vascular changes, leading

to acute inflammation. These products include metabolic acids, extracellular enzymes,

volatile sulphur compounds, lipoteichoic acid and lipopolysaccharides.

Table 2.1 Interactions of plaque bacteria and their products in inflammation
and immunity
Any stimulus that damages host cells or other components will trigger inflammation, and the
resulting inflammation helps activate an immuno-inflammatory response against foreign or
antigenic material present. Conversely, humoral immune reactions will activate an
inflammatory reaction at the site where the antibody binds to the antigen (Williams et al.
1996).

Bacterial products Effects

• activate complement
Whole bacteria • activate neutrophils and macrophages
• are antigenic
Most peptides and proteins secreted by • chemotactic for neutrophils and
bacteria macrophages
• damage host cells
• degrade connective tissue matrix
Enzymes • activate and degrade complement
• degrade antibodies
• are antigenic
• activates complement
• damages some host cells
Lipopolysaccharide (LPS) • activates neutrophils and macrophages
• are antigenic

Polysaccharide plaque matrix and • polyclonal B-cell activator
Bacterial capsule • are antigenic
Other toxins, acids, reducing agents and • damage host cells
metabolites • are antigenic

7

Table 2.1 summarises the interactions of plaque products and their effects on inflammation

and immunity. These factors can directly or indirectly damage sulcular epithelium and

underlying connective tissue, disrupt microvasculature and initiate an inflammatory response

(Darveau et al. 1997). Some aspects of the inflammatory response are clearly distinct but the

precise role played by many of the mediators has not been completely clarified (Page and

Schroeder 1976; Page 1991; Genco et al. 1994; Offenbacher 1996).

2.1 Endogenous mediators of inflammation

The inflammatory exudate flowing from the gingival tissues into the gingival crevice or

periodontal pocket consists of blood components and host defence mediators which can

contain the microbial challenge, or they themselves act as a source of nutrients for the

microbes. The rate of gingival crevicular fluid flow generally reflects the severity of the

inflammation, the increased volume of inflamed tissue and the greater surface area of

pockets (Williams et al. 1996). The initial host response to the bacterial challenge is

characterised by the release of a number of vasoactive and antimicrobial factors:

2.1.1 Histamine:

This mediator of acute inflammation is present in mast cells. Histamine may be released

directly either by:

(a) bacterial mediators such as lipopolysaccharide and enzymes (trypsin like

or proteases) which activate the complement pathway (alternate pathway)

eventually releasing C3a and C5a or

(b) direct complement activation (C3a and C5a), or interleukin-1 and other

factors from endothelial cells, neutrophils and lymphocytes. In addition,

8

antibody-antigen complexes can activate complement (through the classic

pathway) releasing C3a and C5a.

These mediators activate the release of mast cell granules, which increase vascular

permeability (in capillaries and venules), and characteristically are the major mediator of

acute short-lived inflammatory responses.

2.1.2 Bradykinin:

With tissue and vascular injury, serum Hageman factor (Factor XII of the coagulation

cascade) activates the release of bradykinin, a nonapeptide (a long-lived vasodilator)

(Rang et al. 1996; Wilson and Kornman 1996). Bradykinin often follows the release of

histamine and is capable of increasing vascular permeability (Nisengard and Newman

1996). Bradykinin induces:

• continued exudation and crevicular fluid flow

• bone resorption in organ cultures via the prostaglandin cyclo-oxygenase pathway

(Newman et al. 1976; Nisengard and Newman 1996).

2.1.3 Plasmin:

Plasminogen enzyme is a normal constituent of plasma proteins. It is converted to

plasmin by the action of plasminogen activator (also called kallikrein). When the

intrinsic coagulation system is activated, the fibrinolytic system is activated through the

action of plasminogen activators. Activation of Hageman factor (XII) begins a cascade

of reactions in which it catalyses the reaction of circulating plasminogen to plasmin.

Plasmin is a multi-functional protease enzyme that digests fibrin and fibrinogen

(fibrinolysis) and other plasma proteins namely clotting factors II, V, VII and many other

tissue proteins. Plasmin is also an activator of several matrix metalloproteinases (Okada

9

1998). Plasmin derived lysis of the fibrin clot generates fibrin degradation products that

induce vascular permeability and trigger the complement system with the formation of

C3a and C5a components causing the release of histamine from mast cells. These fibrin

products are chemotactic to other inflammatory cells (Walter and Grudy 1993).

2.1.4 Complement:

Specific antibody and complement are two very important antimicrobial factors in GCF.

Activation of complement is one of the first host defences after injury, with these effects:

• vasodilation and increased blood flow (by C2, C3a and C5a)

• activate mast cells to release histamine (by C3a, C5a)

• augment opsonisation (C3b) of bacteria by antibodies and allow some antibodies to

kill bacteria or by phagocytosis

• chemoattractant to neutrophils and macrophages (C3a, C5a) and trigger the release of

prostaglandins, leukotrienes and enzymes into the tissue

• cause pores to open in the membranes of pathogens causing cell lysis (C5-9)

(Dennison and Van Dyke 1997).

Bacteria in the gingival sulcus can activate the complement system via two major

pathways (Page 1991; Offenbacher et al. 1993a):

The classical pathway:

Activation of this system occurs rapidly. This pathway is activated by antigen-

antibody complexes (Dennison and Van Dyke 1997). Complement C1qrs binds

to the Fc component of IgG or IgM antibodies. This is fixed to the bacterial

10

receptor via the Fab region of immunoglobulin activating a cascade of enzymic

reactions to release C3 the precursor of C3A, C3b, C5a, C5b-9 which cause lysis

of cell membranes or functional alterations to promote phagocytosis (Lakhani et

al. 1993; Offenbacher 1996).

The alternative pathway:

Activation of this cascade does not involve immunoglobulin. Activation occurs directly

by bacterial surface lipopolysaccharide (LPS) and endotoxin (from gram-negative

anaerobes). This pathway also involves a series of reactions to release the precursor

complement protein C3 and produce the cleavage products C3a and C5a to C9 (Lakhani

et al. 1993; Offenbacher 1996).

The central event in both pathways is activation or splitting of C3 to C3b which becomes

attached to the activating stimulus (usually bacterial surfaces or antigen-antibody

complexes). Whichever pathway is activated, large amounts of C3a and C3b component

are released, fixing to the inflammatory stimulus and resulting in increased histamine

release, vascular permeability, chemotaxis to phagocytes (promoting phagocytosis), and

promotion of blood clotting. Complement can cause bystander damage since a small

amount may bind to host cells causing lysis or triggering neutrophils to attack.

2.1.5 Platelets:

Platelet adhesion and granule release plays an important role in the early development of the

vascular and cellular aspects of the inflammatory process (Walter and Grudy 1993). Release

of granules from platelets can also help initiate vascular permeability. Mediators released

from platelets include serotonin, a number of coagulation factors and thromboxane A2

(TxA2), all of which are pro-inflammatory. Platelet-derived growth factor (PDGF) is derived

11

from the platelet α−granules that contribute to the repair process (anabolic) following inflam-

matory responses or damaged blood vessels (Walter and Grudy 1993). Other anabolic effects

of PDGF are down regulation of alkaline phosphatase and promotion of proliferation of fi-

broblasts and periodontal regeneration (Okada 1998).

2.2 Eicosanoids

Cellular disturbances (e.g. from cell damage, LPS, complement, thrombin, bradykinin and

antigen-antibody complexes) cause enzymes known as phospholipases to generate

arachidonic acid from the cell membrane phospholipids. Arachidonic acid metabolites are a

small group of lipids known collectively as eicosanoids. Eicosanoids are not found pre-

formed in cells like histamine, they are generated de novo from cell membrane phospholipids.

They control many physiological processes and are the most important mediators and

modulators of the inflammatory reaction (Campbell and Halushka 1996). The prostanoids,

and in particular prostaglandins, are produced from arachidonic acid by cyclo-oxygenase that

occurs in neutrophils, macrophages, mast cells, fibroblasts, lymphocytes, keratinocytes,

osteoblasts and platelets (Offenbacher 1996). Prostanoids encompass all cyclo-oxygenase

products (Table 2.2). The predominant prostanoid product of the inflammatory response in

destructive periodontal diseases is thought to be PGE2 (Howell and Williams 1993).

Table 2.2 Composition of eicosanoids
Eicosanoids
Prostanoids Leukotrienes
All cyclo-oxygenase products
All lipoxygenase products
• prostaglandins

• thromboxane

• prostacyclins

12

2.2.1 General properties of eicosanoids

Eicosanoids are found almost in every tissue and body fluid and have the following

properties:

• they are mediators derived from membrane phospholipids

• they are effector molecules which are formed from polyunsaturated fatty acids

(lipids), mainly arachidonic acid. These include the prostaglandins,

prostacyclins, thromboxane A2 and the leukotrienes.

• their production increases in response to diverse stimuli and they produce a broad

spectrum of biological effects.

• these lipids contribute to a number of physiological and pathological processes

including inflammation, smooth muscle tone, haemostasis, thrombosis,

parturition, and gastrointestinal secretion.

• several classes of drugs, most notably the nonsteroidal anti-inflammatory drugs (and

in particular aspirin), are therapeutically active because they block the formation of

eicosanoids.

(Salmon and Higgs 1987; Davies and MacIntyre 1992; Campbell and Halushka 1996;

Rang et al. 1996).

General effects of prostanoids vary and the type of response elicited is related to specific

target cell receptors (Table 2.2). Their effects are:

i. production of fever, pain and inflammation (Campbell and Halushka 1996).

ii. bone resorption by PGE's (Davies and MacIntyre 1992)

13

iii. PGE2 stimulates cAMP formation in cells, phospholipase C, and calcium influx in

osteoblasts

iv. PGE's also have insulin-like effects on carbohydrate metabolism and exert

parathyroid hormone-like effects that result in mobilisation of calcium ions from

bone (Campbell and Halushka 1996).

v. stimulation of the release of adrenal steroids (ACTH & growth hormone), and of

erythropoietin from the kidney (Davies and MacIntyre 1992; Campbell and

Halushka 1996).

vi. prostaglandins (PGE2, PGD2, PGA2) and prostacyclins (PGI2) are potent

vasodilators, while PGG2, PGH2 and TXA2 are powerful vasoconstrictors (Campbell

and Halushka 1996).

Figure 2.1 shows the products and pathways of cyclo-oxygenase.

Figure 2.1 Products and pathways of cyclo-oxygenase
(Salmon and Higgs 1987).

14

Table 2.3 Cell sources and actions of prostanoids
(Davies and MacIntyre 1992; Campbell and Halushka 1996).
Receptor
Prostanoid Effect Derived from
type
vasodilatation
inhibition of platelet aggregation
PGD2 DP mast cells
relaxation of gastrointestinal muscle
uterine contraction
myometrial contraction
increase in cytoplasmic calcium ions
PGF2a FP Corpus luteum
vasoconstrictor of pulmonary arteries
and veins
vasodilatation
inhibition of platelet aggregation
renin release
tubular reabsorption of sodium ions
vascular
Prostacyclin IP increase cAMP
epithelium
vasoconstriction
platelet aggregation
bronchial-constriction
increase of cytoplasmic calcium ions

bone resorption
increase in cAMP
increases vasodilation
increases vascular permeability
contraction of bronchial and smooth
muscle
bronchial-dilation most nucleated
EP1, stimulation of intestinal fluid secretions cells
PGE2 EP2 relaxation of gastrointestinal smooth especially
EP3 muscle monocytes
contraction of intestinal muscle and macrophages
inhibition of gastric acid secretion
inhibition of lipolysis
inhibition of autonomic
neurotransmitter release
contraction of uterus
decrease of cAMP in adipose cells

2.3 Role of eicosanoids in periodontal tissues

Prostanoid products in the periodontal tissue are primarily mediators of inflammation

and tissue destruction (Offenbacher et al. 1993b; Offenbacher 1996). In view of the

large number of compounds that belong to the eicosanoid family, this section focuses on

15

the main mediator of periodontal inflammation and tissue destruction, i.e. the

prostaglandins and in particular PGE2. In periodontal tissues the actions of PGE2 induce

(Birkedal-Hansen 1993; Offenbacher et al. 1993b):

• vasodilation and increased vascular permeability in the gingival plexus.

• matrix-metalloproteinases (MMP) secretion from macrophages, monocytes and
fibroblasts stimulating connective tissue breakdown.

• increases cAMP in macrophages

• interacts with IL-1 and TNF-α to enhance their effects.

• modulation of platelet and leucocyte reactivity

• inhibition of T cell proliferation

• lysosomal enzymes release from neutrophils

• generation of toxic oxygen radicals from neutrophils

• histamine release from mast cells.

• inhibition of macrophage/monocyte and lymphocyte activation

• generation and secretion of other cytokines.

• osteoclastic bone resorption i.e. increased severity of periodontal diseases (PGE2 has a

major role in periodontitis as a long-lived potent mediator of bone resorption interfering

with the bone remodelling coupling mechanism between osteoblasts and osteoclasts) (Of-

fenbacher 1996; Wiebe et al. 1996; Gemmell et al. 1997; Page et al. 1997; Schwartz et

al. 1997; Ueda et al. 1998).

2.3.1 Biosynthesis of eicosanoids.

The main source of the eicosanoids is arachidonic acid, a 20-carbon polyunsaturated fatty

acid found in the phospholipids of cell membranes and to a lesser extent, in the

glycerides of cell membranes (Davies and MacIntyre 1992). The initial and rate-limiting

16

step to eicosanoid production is the liberation of arachidonate from the membranes

(Rang et al. 1996), either by a one-step process involving phospholipase A2 (PLA2)

directly or the indirect two step process involving either phospholipase C and

diacylglycerol lipase or phospholipase D.

Phospholipase D is an important signal transducer that induces phagocytosis by

phagocytic cells. There are intracellular and extracellular forms of phospholipase A2. It

is mainly the intracellular form that is implicated in the generation of inflammatory

mediators; it generates arachidonic acid and platelet activating factor (PAF), another

powerful mediator of inflammation (Campbell and Halushka 1996; Rang et al. 1996).

The anti-inflammatory action of the glucocorticoids (adrenal hormones e.g. steroids) is

mainly due to the fact that they inhibit the formation of PLA2, inhibiting the induction of

cyclo-oxygenase within the cell and thus reducing free arachidonic acid (Campbell and

Halushka 1996; Rang et al. 1996).

2.3.2 Arachidonic acid pathways: eicosanoid production

There are three pathways for synthesis of eicosanoids from arachidonic acid (Campbell

and Halushka 1996; Sharma and Sharma 1997).

Pathway 1

This involves COX (also known as prostaglandin synthetase). Many stimuli acting on

different cell types can liberate arachidonic acid, for example:

•thrombin on platelets

•C5a on neutrophils

•bradykinin on fibroblasts

•antigen-antibody reactions on mast cells F

17

Free arachidonic acid is metabolised by COX to generate the endoperoxide products
(PGG2/PGH2) which are unstable at normal physiological pH and temperature and are
pivotal in the formation of other products (Salmon and Higgs 1987; Davies and
MacIntyre 1992). These products are either:

•enzymatically converted into either prostaglandins, prostacyclins or
thromboxanes (collectively called prostanoids).

•converted to hydroxy fatty acid (HHT) and malondialdehyde (MDA) by
enzymatic or non-enzymatic pathways (Salmon and Higgs 1987; Davies and
MacIntyre 1992).

COX is bound to the endoplasmic reticulum and primarily has two functions:

•to produce cyclic endoperoxide PGG2

•to convert PGG2 to another cyclic endoperoxide PGH2.

The next steps in arachidonate metabolism vary according to the cell-type secreting
various mediators, each eliciting different physiological functions (Fu et al. 1990; Rang
et al. 1996):

•platelets only produce thromboxane A2 mediator

•vascular endothelium produces prostacyclin mediator

•macrophages/monocytes, fibroblasts produce PGE2

•mast cells produce PGD2

The principal source of PGE2 in periodontal tissues is from macrophages/monocytes and

fibroblasts (although most nucleated cells can produce PGE2) (Fu et al. 1990). There are

two mechanisms whereby PGE2 is produced by macrophages/monocytes.

(a) Bacterial LPS induced PGE2 release

LPS will bind to LBP (a LPS binding protein found in serum) forming a complex

which binds to the high affinity CD14 receptor of macrophages/monocytes,

triggering high intracellular cAMP levels (with very low levels of LPS). This

18

stimulates the release of PGE2, TNF-α and IL-1β. Bacterial antigen-antibody

(IgG) or C3b can elicit the same reaction (Offenbacher et al. 1993b; Offenbacher

1996).

(b) Host induced PGE2

TNF-α and IL-1β have an autocrine effect on the secretory

macrophage/monocyte and a paracrine effect on the residential fibroblast cells

which elicit PGE2, perpetuating the inflammatory response and activating an

immune response.

COX exists in two forms:

•COX-1, a constitutive enzyme found in all cells i.e. it is always present at a constant

concentration in cells but may increase by 2-4 fold upon physiological stimulation,

producing low levels of mediators that are necessary for the maintenance of normal

tissue integrity and function. COX-1 produces the prostanoids (PGI2/6-keto-PGF1α &

TXB2) that regulate normal homeostasis (Sharma and Sharma 1997; Dubois et al. 1998).

•COX-2 is the pro-inflammatory enzyme that is induced by inflammatory stimuli only;

its activity increases 10-80 times following injury or insult. Inflammatory stimuli (eg

LPS) or ligands (eg cytokines) bind to inflammatory cells and eventually induce the

prostanoid mediators of inflammation (Seibert and Masferrer 1994; Seibert et al. 1994;

Gierse et al. 1995; Sharma and Sharma 1997; Dubois et al. 1998).

There are approximately 10 prostaglandins; all have a cyclopentane ring (five carbon

ring) between carbon 8-12 (Figure 2.2). Prostaglandins are named alphabetically from A-

J, with three members in each group (except PGI). These are numbered 1, 2 or 3

(representing the double bonds on the prostaglandin molecule). For example PGE2 has

19

two double bonds between carbon 5-6, and 13-14. The prostacyclins have only two

members, (PGI2 and PGI3).

The thromboxanes (Tx) are closely related to the prostaglandins and are synthesised from

PGH2 (Figure 2.2). These molecules contain an oxane ring (a six carbon ring with an

oxygen atom) instead of a cyclopentane ring. Thromboxane A2 (TxB2) is a potent

vasoconstrictor and triggers platelet aggregation, causing thrombus formation (Sharma

and Sharma 1997; Dubois et al. 1998).

Figure 2.2 The chemical structures of PGE2 and TxB2
(Davies and MacIntyre 1992).

20

Pathway 2

The leukotrienes (LOX)

The second pathway for arachidonic acid metabolism is via the lipoxygenase (LOX)

pathway (Figure 2.1) to provide the parent molecule hydoperoxyeicosatetraenoic acid

(HPETE). The HPETEs are then further metabolised to leukotrienes, hepoxilins,

trioxilins and lipoxins (Sharma et al. 1997). To date there are six HPETEs (5,8,9,11,12

and 15-HPETEs) and each is formed by its corresponding enzyme (Sharma 1997).

Lipoxygenases are soluble enzymes found in the cytosol of cells of lung, platelets, mast

cells and leucocytes. The main enzyme in this group is 5-lipoxygenase; it converts 5-

HPETE to leukotrienes, 12-LOX converts 12-HPETE to hepoxilins and trioxilins, 15-

LOX converts 15-HPETE to lipoxins. Lipoxygenases differ in their specificity according

to the hydroperoxy group (-OOH) on arachidonic acid, and tissues differ in the

lipoxygenase(s) that they contain. For example, platelets have only 12-lipoxygenase and

synthesise 12-(HPETE) whereas leucocytes contain both 5-LOX and 12-LOX and

produce both 5-HPETE and 12-HPETE (Rang et al. 1996) (see Figure 2.1). Arachidonic

acid is enzymatically reduced to hydroxy acids (HPETEs).

The HPETEs are unstable intermediate metabolites (like PGG2 or PGH2) and are further

metabolised by a variety of enzymes. In the leukotriene pathway 5-HPETE is

enzymatically to converted leukotriene-A4 (LTA4) which is unstable, but pivotal in the

formation of other leukotrienes. LTA4 is enzymatically hydrolysed to LTB4 or non-

enzymatically to di-hydroxy acids (di-HETEs). Additionally, LTA4 can be converted

directly to the precursor of cysteinyl-leukotriene LTC4, which is further metabolised to

LTD4, LTE4, and LTF4. LTB4, LTC4, and LTD4 are also known as the "slow reacting

21

substance of anaphylaxis" (SRS-A) (Lewis et al. 1990; McMillan et al. 1992; Salmon et

al. 1987; Snyder et al. 1989).

LTB4, LTC4, and LTD4 are the most potent leukotrienes. LTB4 is a powerful chemotactic

agent for neutrophils and macrophages (acting in picogram amounts) and are important

in the early stages of inflammation. It causes up-regulation of membrane adhesion

molecules of neutrophils, increasing the production of toxic oxygen products and the

release of granule enzymes. It can stimulate proliferation and/or cytokine release from

macrophages (Abramson et al. 1989; Lewis 1990; Rang 1996; Samuelsson 1983).

Arachidonic acid or other polyunsaturated fatty acids may be further metabolised by

lipoxygenases to other oxygenated derivatives of polyunsaturated fatty acids (Rang

1996). A recent addition to these compounds is the lipoxins which were first isolated

in 1984 (Serhan et al. 1984) and generated from within various cells or during cell-cell

interactions. Lipoxins are generated from one of three pathways which can operate

independently or simultaneously (Serhan 1997).

(a) A 15-LOX initiated pathway:

This enzyme is found in eosinophils, macrophages, monocytes and epithelial

cells, under cytokine (IL-1β, TNF-α) and LPS control and regulated by IL-4

and IL-13 (two anti-inflammatory cytokines) (Levy et al. 1993; Nassar et al.

1994; Serhan 1997). Once these cells are stimulated, arachidonic acid is

converted to 15-HPETE or 15-HETE in the donor cell which serve as a

substrate for 5-LOX in the recipient cell (generally neutrophils) which is

converted to lipoxins (by transcellular metabolism) causing vasodilation,

leucocyte regulation and blocking leukotriene metabolism (Serhan 1997).

22

(b) 5-LOX initiated pathway:

This is generally a platelet-neutrophil interaction. This pathway involves 5-LOX

within neutrophils which converts arachidonic acid to 5-HPETE to LTA4 and

platelet 12-LOX induces lipoxin biosynthesis (Romano et al. 1993; Romano et

al. 1992; Serhan 1997).

(c) Aspirin-triggered lipoxins (ATLs).

Aspirin has the ability to irreversibly inhibit COX-1 and COX-2 by acetylating an

essential serine residue site in both enzymes. The acetylated COX enzymes

cannot produce prostaglandins, however more recent medical research shows that

acetylated COX-2 in endothelial or epithelial cells converts arachidonic acid to

15-HETE (Claria et al. 1995; Claria et al. 1996). The 15-HETEs are released

from these cells by cell to cell adherence i.e. to leucocytes (especially

neutrophils) and further metabolised via transcellular pathways by 5-LOX of

leucocytes to form 15-epimeric-lipoxin (15-epi-LX) metabolites (Claria 1995).

The 15-epi-LX metabolites are also termed aspirin triggered lipoxins (ATLs).

Endothelial and epithelial cell COX-2 when induced by pro-inflammatory

cytokines (IL-1β, TNF-α) and LPS in the presence of aspirin can shunt

arachidonic metabolism to synthesise 15-epi-LX molecules. The 15-epi-LX

molecules serve as “stop signals” (i.e. to evoke anti-inflammatory effects)

causing vasodilation, inhibiting neutrophil adhesion to endothelial cells

(diapedesis), chemotaxis and cell proliferation (Claria 1996; Claria 1995; Clish et

al. 1999; Serhan 1997; Takano et al. 1997).

23

From these recent findings it may be that lipoxin production may be an important

anti-inflammatory event, especially by ATLs. However most studies on lipoxins

use in vitro or in vivo models. However further research is needed to understand

the biofeedback regulatory mechanisms involved in converting from a pro-

inflammatory eicosanoid phenotype to an anti-inflammatory phenotype.

Currently there is no research on lipoxin (especially ATL) involvement in the

gingival or periodontal inflammatory process. Nevertheless ATLs may be a

further anti-inflammatory (beneficial) pathway provided by aspirin.

Pathway 3

This pathway involves the cytochrome P450 group of enzymes (present in endoplasmic

reticulum) breaking down arachidonic acid to HETEs and DiHETEs.

The predominant pathways in eicosanoid production are pathways I and 2 (Sharma and

Sharma 1997).

2.3.3 Catabolism of the eicosanoids

A number of intra-cellular enzymes are involved in the catabolism and inactivation of

most eicosanoids. There are prostaglandin-specific enzymes that rapidly inactivate the

prostaglandins and their metabolites are excreted in the urine. About 95% of PGE2,

PGE1 and PGF2a are inactivated during their first passage through the pulmonary

circulation. The half-life of most prostaglandins is less than a minute in the circulation

(Campbell and Halushka 1996; Rang et al. 1996). Leukotriene products are inactivated

by oxidative pathways or degraded in the kidneys, lungs and liver (Campbell and

Halushka 1996).

24

2.4 The role of cytokines in periodontal tissues

Table 2.4 shows the major mediators thought to be involved in the pathogenesis of

periodontitis (Schenkein 1999). Arachidonic acid metabolites have been discussed in

section 2.3, the next section only discusses the interleukins and Tumour Necrosis Factor

alpha (TNF-α). All these mediators primarily interact with each other and have impact

on the pathogenesis of periodontal diseases, but prostaglandins are the major mediators

involved in tissue destruction in conjunction with the following cytokines.

The term cytokine means “cell protein”. Cytokines direct and regulate inflammation and

wound healing (Page et al. 1997; Okada 1998). Subgroups of cytokines are:

• the interleukins which carry complex and detailed messages between leucocytes,

• the growth factors which trigger myelopoesis, leucocyte mitosis and cell

differentiation

• chemokines which trigger cell recruitment

• interferons, lymphocyte activating molecules (Birkedal-Hansen 1993; Offenbacher

1996).

Cytokines, lymphokines and monokines have autocrine (self-regulate the cells producing

the cytokine), paracrine (modulate distant cells not producing the cytokine) and

intracrine (actions within a cell) effects on target cells (Okada 1998). All cytokines

activate target cells by binding to specific receptors on their cell membranes. This

receptor-ligand coupling triggers cellular activation of the target cell, modifying the cell's

activity (Birkedal-Hansen 1993; Offenbacher 1996), e.g. IL-1 binds to fibroblasts to

trigger the release of collagenase to degrade collagen in the immediate environs. Often

inflammatory cytokines trigger the secretion of specific enzymes, lipids, bioactive

25

amines and reactive oxygen metabolites that serve as effector molecules (Alexander and

Damoulis 1994). Periodontal tissue destruction is via mobilisation and activation of

macrophage/monocytes, lymphocytes and fibroblasts. The modulation of these events is

via catabolic cytokines and inflammatory mediators.

Table 2.4 Major tissue destructive mediators in periodontitis
Interleukins (1, 6, 8)
Tumour Necrosis Factor alpha (TNF-α)
Arachidonic Acid Metabolites (PGE2)

Interleukin-1 (IL-1)

Interleukin-1 is a polypeptide, which has diverse roles in immunity, inflammation, tissue

breakdown and homeostasis. It is synthesised by macrophages, monocytes,

lymphocytes, endothelial cells, fibroblasts, keratinocytes and brain cells. In the

periodontal tissues, macrophages predominantly secrete IL-1, Il-1α and Il-1β. Both

forms bind to the same cell receptors of many cell types and in various densities

(Alexander and Damoulis 1994; Offenbacher 1996; Mathur and Michalowicz 1997;

Soskolne 1997; Ellis 1998; Okada 1998; Schenkein 1999)

Properties of IL-1:

• increases adhesion molecules on fibroblasts, immunocytes (stimulates

proliferation of keratinocytes and endothelial cells)

• enhances fibroblast synthesis of collagenase, fibronectin and PGE2

• induces the production of matrix metalloproteinases (MMPs) in

periodontitis.

• elevates the levels of pro-collagenase in gingival and periodontal ligament

fibroblasts.

• stimulates plasminogen activator in gingival fibroblasts, resulting in the

26

generation of plasmin which is a naturally occurring activator of several

matrix metalloproteinases.

• activates both T- and B-cells. It also promotes B-cell activation,

proliferation, clonal expansion and antibody secretion. It “primes”

macrophages and neutrophils by up-regulating receptors for complement

and immunoglobulins.

• T-cells regulate the immune response by increasing or decreasing IL-1

secretion. T-cells release gamma interferon (IFN-γ) which enhances

secretion of IL-1 and PGE2 from LPS-stimulated macrophages. Therefore

IFN-γ serves to up-regulate the inflammatory response. (Page 1991;

Birkedal-Hansen 1993; Alexander and Damoulis 1994; Mathur and

Michalowicz 1997; Soskolne 1997; Ellis 1998; Okada 1998; Page 1998)

• IL-1β and IL-1α are potent connective tissue catabolic stimulators. They

directly stimulate bone resorption, and trigger the release of PGE2 from

fibroblasts and macrophage/monocytes. PGE2 (Seymour et al. 1993;

Tatakis 1993).


IL-6 influences immune and inflammatory responses and the main sources are from

stimulated fibroblasts, endothelial cells, macrophages, T and B-cells and keratinocytes.

IL-6 shares many biological properties with IL-1 and has been found to:

• be in higher concentrations levels in inflamed sites than healthy sites

• stimulate eicosanoid production

• stimulate MMP production

27

• stimulate B-cells into Ig-secreting plasma cells.

• be a potent stimulator of IgG1.

• plays a major role in regulating bone turnover and is essential for bone loss

caused by oestrogen deficiency (menopause)

• act as a paracrine and/or autocrine factor in bone resorption in pathologic

states, by stimulating osteoclasts and activating bone resorption (Page 1991;

Alexander and Damoulis 1994; Offenbacher et al. 1996; Mathur and

Michalowicz 1997; Soskolne 1997; Ellis 1998; Okada 1998; Schenkein

1999).


Produced by leucocytes and keratinocytes in response to LPS, IL-1 or TNF-α, with the

following properties:

• proinflammatory

• strong chemoattractant to neutrophils.

• selectively stimulate MMP in macrophages keratinocytes

Local tissue destruction in periodontitis or inflamed gingiva may be due to the

continuous and excessive IL-8 levels that in turn mediate chemotactic and activation

effects on neutrophils and production of MMPs. IL-8 may also attract and induce T-cell

proliferation (Alexander and Damoulis 1994; Mathur and Michalowicz 1997; Soskolne

1997; Ellis 1998; Okada 1998).

Tumour necrosis factor α (TNF-α)

This proinflammatory cytokine is mainly secreted by monocytes and macrophages; it has

the following properties:

28

• induces secretion of collagenase by fibroblasts

• induces resorption of cartilage and bone

• induces periodontal tissue breakdown in periodontitis

• in resting macrophages it induces synthesis of IL-1 and PGE2

• activates osteoclasts and thus induces bone resorption although both forms
of IL-1 are at least 10 times more potent on a molar level than TNF-α in the
induction of bone demineralisation

• has synergistic effects with IL-1 in bone resorption actions

• lipopolysaccharide (LPS) from gram negative bacteria can initiate the
production of TNF-α from macrophages/monocytes (Alexander and
Damoulis 1994).

Periodontal homeostasis represents a delicate balance between anabolic and catabolic

activities (Offenbacher 1996). Myriads of cytokines are involved in tissue turnover and

the maintenance of the integrity of the periodontium; of interest are the interleukins,

prostaglandins, interferons and colony stimulating factors which mediate inflammatory

and immune responses (Williams et al. 1996). Cytokines in association with PGE2 are

thought to lead to alveolar bone resorption, inhibition of bone formation and synthesis of

collagenase by gingival fibroblasts which degrades matrix collagen (Page 1991;

Alexander and Damoulis 1994; Offenbacher et al. 1996; Mathur and Michalowicz 1997;

Ellis 1998; Okada 1998; Ueda et al. 1998).

One of the significant advances in periodontal research in the last 20 years has been the

finding that normal residential cells of the periodontium can be induced to a catabolic

state by exposure to LPS, IL-1, TNF-α and PGE2 and participate in tissue destruction

(Reynolds and Meikle 1997; Schwartz et al. 1997). In periodontal health, fibroblast

genes for collagen synthesis and TIMPs are turned on while the genes for MMPs are

turned off. During periodontitis, the reverse applies, with fibroblasts producing also

29

IL-1β. This cytokine may cause autocrine stimulation with more IL-1β being secreted,

or affect other target cells such as monocytes/macrophages, epithelial and endothelial

cells (paracrine stimulation) to further enhance the production of the MMPs and PGE2

(Reynolds and Meikle 1997; Schwartz et al. 1997; Schenkein 1999).

Clinically healthy gingival and periodontal tissues express a number of anabolic growth
factors:

• epidermal growth factor (EGF)

• platelet-derived growth factor (PDGF)

• transforming growth factor (TGF-β) - this is a superfamily of proteins and
contains a number of bone morphogenic proteins (BMPs)

• insulin-like growth factor (IGF)

• cementum-derived growth factor (CGF)

• inflammatory cytokines such as (IL-1, IL-6, and TNF-α) are in low
concentrations compared to inflamed sites

These anabolic molecules are involved in the rebuilding of the extracellular matrix by:

• chemoattracting fibroblasts, periodontal ligament cells and bone generating
cells

• stimulating cells from a stable (nondividing) cycle to undergo mitosis and
thus increasing the number of stromal cells

• inducing cell differentiation of connective tissue mesenchymal cells to
matrix secreting cells (Bartold et al.1998).

Many of these molecules are incorporated into newly formed extracellular matrices,

which they induce. During wound healing or repair, macrophages are drawn to these

sites by clotting factors. These macrophages respond differently than the pro-

inflammatory macrophages which are chemo-attracted to sites by bacterial products or

complement products (Bartold et al.1998).

30

2.5 Cellular events in inflammation

The acute inflammatory response is characterised by the presence of neutrophils

(Miyasaki 1991; Miyasaki et al. 1994; Miyasaki 1996) which constitute approximately

90% of total circulating leucocytes and are the body's first line of defence against

microbes (Van Dyke and Hoop 1990). Neutrophils have at least three types of

cytoplasmic membrane-enclosed granules: primary granules or azurophil granules,

secondary granules or specific granules and tertiary or secretory granules (Table 2.5).

These granules can release a large variety of enzymes that can degrade host tissue

(collagenase, elastase, β-glucuronidase); this is a part of normal tissue homeostasis

resulting in remodelling or healing. In addition these granules have a large number of

antimicrobial substances which can kill ingested microorganisms once phagocytosed.

Table 2.5 Neutrophil components and function (Williams et al. 1996).

Granule Function
(all function under anaerobic and aerobic conditions)
Granule component Effect
Primary granules Cellular myeloperoxidase Microbial killing
(azurophil) Lysosome
Cationic proteins Histamine release +
enhances phagocytosis
Acid hydrolases Antibacterial
β−Glucoronidase
α−Μannoxidase
Neutral protease
Elastase Exacerbates and mediates in
inflammation
Cathepsin
Secondary (specific) Lysosome Hydrolysis of cell wall
granules Alkaline phosphatase proteoglycans
Collagenase Collagen degradation
Vitamin B12 binding
proteins
Lactoferrin Bactericidal
Tertiary (secretory) Gelatinase Replenish cell surface
granules Alkaline phosphatase receptor expression and
adhesion

31

2.5.1 Macrophage phenotypes

Since the host defences cannot inactivate biofilm completely, the inflammatory response

in periodontitis is longstanding and chronic. The presence of the macrophage signals

chronicity; the neutrophil/macrophage characterises chronic inflammation while the

presence of macrophage/lymphocytes characterises the immune response (Williams et al.

1996). The distinction between chronic inflammation and immunity is not that clear-cut.

Macrophages have an important role in antigen processing as parts of the development of

an immune response and a subset of these cells have phagocytic capacity (Page et al.

1997). Macrophages arise from bone marrow in a functional, immature condition, but

eventually differentiate in the tissues. Macrophage phenotypes may phagocytose

bacteria, modulate the clearance of damaged tissue debris during inflammation, modulate

tissue remodelling, and trap and present antigens to lymphocytes (helper T-cells) to

induce the immune response. (Miyasaki 1996; Page et al. 1997).

Macrophages are capable of synthesizing cytokines that contribute to healing and repair

(anabolic) but can also have pro-inflammatory effects (catabolic) in the presence of a

chronic microbial challenge. (Page 1991; Seymour 1991; Birkedal-Hansen 1993;

Offenbacher et al. 1993b; Genco et al. 1994). Macrophages represent 5-39% of

infiltrating cells in inflamed periodontal tissues (Toppal et al. 1989; Zappa et al. 1991;

Okada 1998).

The macrophage is the key cell in directing whether anabolic or catabolic changes occur

within the periodontal tissues. The catabolic changes occurring in the gingival and

periodontal tissues are due to the presence of the proinflammatory macrophages which

have distinctive properties:

32

• reactive oxygen metabolites, including the superoxide anion(O2-), hydrogen
peroxide (H2O2), the hydroxyl ion (OH-), and hypochlorous acid (HOCl-). All
are bactericidal but can also be toxic to host cells (Klebanoff 1992; Alexander
and Damoulis 1994).

• arachidonic acid metabolites such as PGE2 and LTB4 and these can be
produced in large amounts to create an inflammatory reaction (Samuelsson
1983; Salmon and Higgs 1987; Davidson 1992; Offenbacher et al. 1993b).

• secreting the proinflammatory cytokines IL-1, IL-6, and TNF-α (Offenbacher
1996)

In the periodontium, macrophage PGE2 has many regulatory effects:

• decrease adherence and migration of macrophages,

• under the influence of IL-1β and TNF-α , inhibits the genes controlling the
synthesis of collagen and non-collagenous matrix proteins and tissue
inhibitors of metalloproteinases (TIMPs) in fibroblasts

• stimulates the synthesis and release of matrix metalloproteinases (e.g.
collagenase)

• it is the major mediator of pathological bone resorption

• suppresses leucocyte function

• along with cytokines IL-1, TNF-α and interferon-γ (INF-γ) it can regulate IgG
production (where high concentrations of PGE2 inhibit antibody production
and low concentrations act synergistically with IL-4 and enhance IgG
production (Miyasaki 1996; Page et al. 1997; Reynolds and Meikle 1997).

2.5.2 Alveolar bone resorption

Knowledge about alveolar bone resorption has lagged behind the understanding and

research on connective tissue breakdown. There are, however, some well established

facts about bone remodelling and resorption (Schwartz et al. 1997):

33

• bone resorption-formation is a tightly coupled process and PGE2, IL-1, TNF-α
are known mediators of bone loss.

• IL-6 mediates the formation osteoclasts to resorb bone

• activities between osteoblasts, osteoclasts and osteocytes are highly integrated
and coordinated with one another.

• there are biofeedback loops between these cells, where osteoblasts produce
local factors that induce osteoclastic activity and vice versa.

• cell control is regulated by circulating factors such as steroid hormones,
parathyroid hormone, calcitonin and vitamin D.

More information is needed to fully understand this area of periodontal pathogenesis.

This overview of the inflammatory response is brief; the reaction of the inflammatory

response is very complex, and is integrated with the immune system. The aim of this

review was to show the major inflammatory pathways and their roles in the overall

pathologic process.

2.6 Nonsteroidal anti-inflammatory drugs in periodontal diseases.

The main anti-inflammatory agents are the glucocorticoids and the non-steroidal anti-

inflammatory drugs (NSAIDs). Most of these drugs have anti-inflammatory, analgesic

effects and antipyretic effects which are related to their inhibition of the actions of COX

(Vane 1971) and thus the inhibition of prostaglandins and thromboxanes (Heasman and

Seymour 1989; Heasman et al. 1989; Williams et al. 1989; Heasman et al. 1990;

Czuszak et al. 1996).

2.6.1 History of salicylates

Aspirin (acetylsalicylic acid) is the most widely used medicinal agent in the Western world

(Vane et al 1992; Rainsford 1994). Natural products that contain precursors of salicylic acid,

such as willow bark (which contains the glycoside salicin) and oil of wintergreen (which con-

34

tains methylsalicylate) have long been used in the treatment of rheumatism. (Walton et al.

1994). Since the early 1800’s, salicin was hydrolysed to glucose and salicylic alcohol, which

was then converted to salicylic acid. Sodium salicylate was first used in 1875 in the treatment

of rheumatic fever and as an anti-pyretic. The success of this drug prompted Hoffman, a

chemist employed by Bayer, to prepare acetylsalicylic acid based on the earlier, but forgotten,

work of Gerhardt in 1853. The name aspirin is derived from Spiraea, the plant species from

which salicylic acid was once prepared (Campbell and Halushka 1996). According to Rains-

ford (Rainsford 1984) the Bayer Company enjoyed immense profitability from aspirin by pro-

tecting its patents until the beginning of World War I. This was when other companies started

to process aspirin and challenged the Bayer monopoly. In USA the patent office cancelled

Bayer’s registered rights to the name of aspirin, in 1918 and the US Supreme Court ruled that

there was no infringement of tradename rights by US companies because Bayer’s aspirin had

been over-advertised to such an extent that it had become a common name. At about the

same time in Australia, the Federal government also suspended the Bayer’s patent rights. A

pharmacist George Nicholas with a chemist called Smith produced the first Australian aspirin

by 1915. Smith later withdrew from the partnership. George Nicholas joined with his brother

and formed the Nicholas Proprietary Ltd. They registered their aspirin under the trademark of

“Aspro” (Rainsford 1984).

2.6.2 Physio-chemical properties of aspirin and other salicylates .

The structure of aspirin is shown in Figure 2.3 (Campbell and Halushka 1996). Aspirin is

rapidly absorbed from the gastrointestinal tract, partly from the stomach and mainly from the

upper small intestine where it is quickly hydrolysed to salicylate (salicylic acid) by esterase

enzymes in the gut wall, blood and liver.

35

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