8-Isoprostane: A lipid peroxidation product in gingivalcrevicular fluid in healthy, gingivitis and chronicperiodontitis subjects

A.R. Pradeep *, Nishanth S. Rao, Pavan Bajaj, Esha Agarwal

Department of Periodontics, Government Dental College and Research Institute, Bangalore 560002, India

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a r t i c l e i n f o

Article history:

Accepted 15 January 2013

Keywords:

Chronic periodontitis

F2-isoprostanes

Oxidative stress

a b s t r a c t

Objective: The idea that reactive oxygen species (ROS) are associated with the pathogenesis

of inflammatory periodontal diseases and have a role (direct or indirect) in tissue damage

has become a major area of research over the last decade. The purpose of this study is to

determine, presence of 8-isoprostane in gingival crevicular fluid (GCF) in healthy, gingivitis

and chronic periodontitis (CP) subjects and to find an association, if any between GCF 8-

isoprostane levels and clinical periodontal parameters.

Materials and methods: 78 subjects (40 males and 38 females) were selected based on their

clinical parameters into three groups: Group 1 (26 healthy), Group 2 (26 gingivitis subjects)

and Group 3 (26 CP subjects). GCF 8-isoprostane levels were estimated by ELISA.

Results: The 8-isoprostane concentration in GCF was highest in subjects with chronic

periodontitis as compared to gingivitis and healthy subjects and a significant association

was observed between GCF 8-isoprostane levels and all periodontal parameters.

Conclusions: There was increase in 8-isoprostane levels in GCF as the disease process

progressed from health to gingivitis and chronic periodontitis, suggesting a role for in-

creased oxidative stress in CP.

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1. Introduction

Periodontal diseases represent chronic inflammatory

responses to a bacterial challenge. Although bacterial biofilms

have been shown to be necessary in the initiation of gingival

inflammation and subsequent destruction of periodontal

tissues, its presence alone explains a relatively small propor-

tion (i.e., 20–30%) of the variance in disease expression.1,2

Based on an established model of pathogenesis, the bacterial

biofilm alone is insufficient to explain disease initiation and

progression. Evidence suggests that periodontal tissues

destruction is mainly due to the host’s inflammatory response

to the bacterial challenge.3

* Corresponding author. Fax: +91 8026703176.E-mail address: periodonticsgdcri@gmail.com (A.R. Pradeep).

0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.archoralbio.2013.01.011

Within the gingival crevice, neutrophils perform an innate

cellular host defense role. These neutrophils contribute half of

the leucocytes infiltrating the junctional epithelium and 90%

of the leucocytes isolated from crevicular fluid.4 Following the

stimulation by bacterial antigen, neutrophils as well as

macrophages produce O2� (superoxide) and other reactive

oxygen species (ROS) via the metabolic pathway of the

‘‘respiratory burst’’, during the process of phagocytosis.5

ROS serve as agents highly toxic to the internalised microbial

agents; however they can also lead to extracellular structure

damage.5 Further, high total ROS generation by neutrophils

from chronic periodontitis (CP) as compared with that of

control individuals has been demonstrated, thus highlighting

the role of ROS in periodontal destruction.6

d.

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Protection against ROS is provided by antioxidants (AO). In

healthy organisms, the balance is maintained among oxidants

and AO’s but under pathologic conditions, the balance may be

tilted towards the oxidative side causing ‘‘oxidative stress’’.

Oxidative stress is an important physiologic modifier of

immune and inflammatory mechanisms.7

Isoprostanes (IsoP) are prostaglandin (PG)-like compounds

formed from the peroxidation of arachidonic acid.8,9 Unlike

PGs, however, they do not require the cyclooxygenase for their

formation. A substantial body of evidence indicates that

measurement of IsoP in body fluids such as urine, serum or

plasma provides a reliable noninvasive approach to assess

lipid peroxidation in vivo and represents a major advance in

our ability to assess oxidative stress status in animals and

humans.10,11 Plasma 8-iso-prostaglandin F2a (8-epi PGF2a or 8-

IsoP) levels are elevated in patients with atherosclerosis, acute

myocardial infarction, alzheimer’s and other neurodegenera-

tive diseases, rheumatoid arthritis, psoriatic arthritis, reactive

arthritis, osteoarthritis, systemic sclerosis, diabetes mellitus

(DM) and periodontal disease.12

Investigation of peripheral oxidative damage in periodon-

titis is in its infancy but recent reports have suggested

increased levels of protein carbonyls and 8-IsoP in peripheral

blood and saliva in CP subjects.13,14

Gingival crevicular fluid (GCF) is a dynamic fluid that

emerges between the surface of the tooth and the epithelial

integument. It has been recognised for over 100 years, but still

the exact nature of the fluid, its origins and composition, has

been the subject of controversy. This may be a result of

variations in the amount and/or nature of the fluid produced

under different clinical conditions and the use of a wide

variety of sampling methods.15 Gingival crevicular contents

provide a potential source of markers of the destruction of

periodontal structures and the disease activity. The major

attraction of GCF as a diagnostic marker is the site-specific

nature of the sample. This allows laboratory investigations of

GCF constituents to be linked to clinical assessments at the

site of sample selection.

However, till date, 8-IsoP concentration in GCF in chronic

periodontitis (CP) subjects has not been explored. Thus, in

view of the aforementioned findings, this clinicobiochemical

study was undertaken to further probe into the role of

oxidative stress in periodontal diseases by estimating the

GCF levels of 8-IsoP in subjects with clinically healthy

periodontium, in patients with gingivitis and chronic peri-

odontitis.

2. Materials and methods

The study was carried out from April 2011 to July 2011. The

study group consisted of 78, age and gender balanced subjects

(25–45 years; gender: 40 males and 38 females) attending the

outpatient section, Department of Periodontics, Government

Dental College and Research Institute, Bangalore. Written

informed consent was obtained from those who agreed to

participate voluntarily. Patients with aggressive periodontitis,

hypertension, a smoking habit, gross oral pathology, heart

diseases, rheumatoid arthritis, diabetes, tumours, or any other

systemic disease that can alter the course of periodontal

disease, or those who had any course of medication affecting

periodontal status or had received periodontal therapy in the

preceding 6 months were excluded from the study. The Ethical

Clearance was approved by Institutional Ethical Committee

and Review Board. Each subject underwent a full-mouth

periodontal probing and charting, Body Mass Index (BMI)

charting as per WHO guidelines16 and periapical radiographs

were taken using the longcone technique. Only subjects

having BMI in the normal range of 18.5–24.9 kg/m2 and a

minimum of 20 natural teeth were selected in this study.

Radiographic bone loss was recorded dichotomously (pres-

ence or absence) to differentiate patients with chronic

periodontitis from other groups. Subjects were categorised

into three groups based on the gingival index (GI),17 probing

pocket depth (PPD), Clinical attachment level (CAL), and

radiographic evidence of bone loss. Group 1 (healthy)

consisted of 26 subjects with clinically healthy periodontium,

GI = 0 (absence of clinical inflammation), PPD � 3 mm, and

CAL = 0, with no evidence of bone loss on radiographs. Group 2

(gingivitis) consisted of 26 subjects who showed clinical signs

of gingival inflammation, GI > 1, without any attachment loss,

PPD < 3 mm. Group 3 (CP) consisted of 26 subjects who had

signs of clinical inflammation, GI > 1, greater than 30% sites

had PPD � 5 mm, and CAL � 3 mm, with radiographic evi-

dence of bone loss.

2.1. Site selection and GCF fluid collection

All clinical examinations, radiographs, group allocations, and

sampling site selections were performed by one examiner

(ARP), and the samples were collected on the subsequent day

by a second examiner (NSR). This was to prevent contamina-

tion of GCF with blood associated with the probing of inflamed

sites. A calibrated examiner performed all the clinical

assessments using a University of North Carolina (UNC)-15

periodontal probe (Hu Friedy, Chicago, IL, USA), to ensure

adequate intra-examiner reproducibility. Only one site per

subject was selected as a sampling site. In healthy group, to

ensure adequate volume, GCF was pooled from multiple sites

with no inflammatory signs. In gingivitis patients, site with

highest clinical signs of inflammation i.e., redness, bleeding

on probing and oedema in the absence of CAL was selected. In

patients with chronic periodontitis, the site showing the

greatest CAL and signs of inflammation, along with radio-

graphic confirmation of bone loss, was selected for sampling.

After making the subjects sit comfortably in an upright

position on the dental chair, the selected test site was air dried

and isolated with cotton rolls. Without touching the marginal

gingiva, supragingival plaque was removed to avoid contami-

nation of the paper strips (Periopaper, Ora Flow Inc.,

Amityville, NY, USA) using the intracrevicular method

‘superficial’ developed by Loe & Holm-Pederson.18 The

absorbed GCF volume of each strip was determined by

electronic impedance (Periotron 8000, ProFlow Inc., Amityville,

NY, USA). The same method was used to obtain GCF samples

from the control group. The readings from the Periotron 8000

were converted to an actual volume (ml) by reference to the

standard curve.

Two Periopaper strips that absorbed GCF for each subject

were pooled and the Periopaper strips were placed in a sterile

Table 1 – Descriptive statistics of study population(mean W S.D.).

Study group Group 1(n = 26)

Group 2(n = 26)

Group 3(n = 26)

Age (years) 30.70 � 3.683 31.47 � 3.623 30.20 � 3.299

Sex (M/F) 14/12 13/13 13/13

GI – 1.85 � 0.37 2.15 � 0.68

PPD (mm) 1.50 � 0.51 2.54 � 0.51 7.38 � 1.20

CAL (mm) – – 5.85 � 1.22

GCF volume (ml) 0.24 � 0.09 0.42 � 0.21 0.86 � 0.11

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eppendorff vial containing 400 ml of phosphate buffer saline

and kept at �70 8C until analysed. Periopaper strips contami-

nated with blood and saliva were excluded or discarded.

Periodontal treatment (Scaling and Root Planing) was per-

formed for periodontitis patients at the same appointment

after GCF collection.

2.2. 8-IsoP analysis

The samples were then assayed for 8-IsoP using enzyme-

linked immunosorbent assay (ELISA) kit according to manu-

facturer’s instructions (Cayman Chemical Company, Ann

Arbour, MI, USA). The GCF sample tubes were first gently

shaken for 1 min and then centrifuged for 5 min at 1500 g to

elute. The elute was then used as sample for ELISA estimation

from GCF samples. Briefly, samples were incubated in the

wells of a divided microplate that had been precoated with the

Mouse Anti-Rabbit IgG and blocked with a proprietary

formulation of proteins. On incubation with 8-IsoP Tracer,

8-IsoP EIA Antiserum, 8-IsoP was detected in the samples.

After final incubation with Ellman’s Reagent (which contains

the substrate to AChE) absorbance was read on ELISA reader.

The total 8-IsoP was determined in picograms (pg), and the

calculation of the concentration in each sample was per-

formed by dividing the amount of 8-IsoP by the volume of

sample (pg/ml).

2.3. Statistical analysis

The data were analysed using statistical software (SPSS

version 10.5, SPSS, Chicago, USA). Power calculations were

performed before the study was initiated. To achieve 90%

power and detect mean differences of the clinical parameters

between groups, 25 sites per group were required. Analysis of

Variance (ANOVA) and Scheffe’s post hoc analysis was carried

out for a comparison of GCF 8-IsoP levels between the groups.

Using Pearson’s correlation coefficient, the relationship

between GCF 8-IsoP concentration and the clinical param-

eters. p values < 0.05 were considered statistically significant.

3. Results

The descriptive statistics along with the mean � SD of age,

sex, PPD and CAL of the sites of GCF sample collection (of all

groups) are tabulated in Table 1. All the samples in each group

tested positive for 8-IsoP assay. The mean 8-IsoP concentra-

tion in GCF was highest for Group 3 followed by Group 2 and

Table 2 – Results of ANOVA and pair-wise comparison using

groups.

Study groups GCF 8-IsoP conc. (pg/ml) ANOVA

F-Value p-Valu

Group 1 195.65 � 69.27

Group 2 293.27 � 63.67 239.688 <0.001

Group 3 832.81 � 171.17

* Significant at p value < 0.05.

least in Group 1. (Table 2) When Groups 1 and 2, 1 and 3, 2 and 3

were compared, the differences in the mean GCF 8-IsoP

concentrations were statistically significant. (Table 2) Pearson

correlation coefficient between the clinical parameters and 8-

IsoP levels are tabulated in Table 3. The correlation between 8-

IsoP levels & PPD was found to be significantly positive in all

the groups. However, the correlation between 8-IsoP levels &

GI was found to be significantly positive only in Group 2 and

weak positive (not statistically significant at p value > 0.05)

correlation in Group 3. The correlation between 8-IsoP levels

and CAL was found to be positive and statistically significant

( p > 0.05) in Group 3.

4. Discussion

The idea that ROS are associated with the pathogenesis of a

variety of inflammatory diseases and have a role (direct or

indirect) in tissue damage has become a major area of research

over the last decade and recently oxidative stress induced

bone loss has been implicated in periodontitis.19

Current evidence indicates that periodontal disease occurs

in predisposed individuals with an aberrant inflammatory/

immune response to microbial plaque. Although all cells

produce ROS during normal physiological functions,20 it is

mononuclear and neutrophilic polymorphonuclear phago-

cytes that produce high levels to facilitate the killing and

destruction of microbes.21 Plaque bacteria and their products

are an obvious source of factors that could stimulate

neutrophils infiltrating the periodontal tissues.22 Enhanced

ROS generation by peripheral neutrophils from patients with

both chronic and aggressive disease can be stimulated with

opsonized bacteria associated with periodontal disease.22,23

This finding suggests that the hyperreactive phenotype of

peripheral neutrophils could have local tissue-damaging

consequences.24

Scheffe’s test of the mean GCF 8-IsoP Conc between three

Scheffe’s test

e Mean difference (I�J) p-Value

Group 1 vs group 2 �97.615* 0.003*

* Group 1 vs group 3 �637.154* <0.001*

Group 2 vs group 3 �539.538* <0.001*

Table 3 – Relationship of GCF 8-IsoP levels to clinicalparameters.

Parameters Group 1 Group 2 Group 3

GI – 0.466* 0.353

PPD 0.777* 0.766* 0.937*

CAL – – 0.815*

* Significant at p value < 0.05.

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Numerous studies have demonstrated an increase in

products of oxidative damage in peripheral blood from

periodontitis subjects compared with healthy individuals.13,25

Most methods available to assess oxidant stress in vivo

previously have suffered from a lack of sensitivity and/or

specificity or are unreliable. However, a substantial body of

evidence indicates that measurement of IsoP in body fluids

such as urine or plasma provides a reliable noninvasive

approach to assess lipid peroxidation in vivo and represents a

major advance in our ability to assess oxidative stress status in

animals and humans.10,11 A number of studies have been

reported examining the utility of quantifying IsoP as an index

of oxidant stress in association with human disease.10,11,14

Our study comprised of three groups (healthy, gingivitis

and chronic periodontitis subjects), these groups helped us to

evaluate the role of 8-IsoP in periodontal disease progression.

The results of the present study are in accordance with that of

a previous study by Wolfram et al. who found that elevated

salivary 8-IsoP levels, is associated with periodontal disease

and is significantly aggravated by concomitant tobacco

abuse.14 The results of the present study indicated that

concentration of 8-IsoP in GCF increased progressively from

healthy (195.65 � 69.27) to gingivitis (293.27 � 63.67) and

periodontitis (832.81 � 171.17) sites, suggesting that oxidative

stress increases as the periodontal disease advances from

health to gingivitis and is much higher in chronic periodontitis

subjects.

In the present study, 8-IsoP levels were estimated in GCF

collected using the absorbent filter paper strips. The advan-

tages of the technique are that it is quick and easy to use, can

be applied to individual sites and, possibly, is the least

traumatic when correctly used. The electronic measuring

device, the Periotron, allowed accurate determination of the

GCF volume and subsequent laboratory investigation of the

sample composition. Also the analysis of 8-IsoP levels in GCF

would provide a better picture on its role in the pathogenesis

of periodontitis as compared to its estimation saliva and

serum. The variability of 8-IsoP concentration within the

patients of each group can be attributed to the role of 8-IsoP in

different stages of disease process at the time of collection of

GCF and serum samples.

One of the drawbacks of the current study was that the

levels of anti-oxidants were not determined along with the

levels of 8-IsoP. The levels of anti-oxidants would have given a

better picture of the true oxidative stress experienced by the

chronic periodontitis subjects. Future studies may be carried

out bearing this in perspective to determine the role of 8-IsoP

in chronic periodontitis.

The 8-IsoP levels in GCF in the current study was found to

significantly correlated with GI, PPD and CAL suggesting that

its levels increase the as the disease progresses. Thus further

research on a possible prognostic role for 8-IsoP and its use for

estimating disease progression need to be carried out. Thus

the results of our study seem to suggest that 8-IsoP may be a

marker for oxidative stress in chronic periodontitis, however

this hypothesis needs further testing.

5. Conclusion

Thus within the limits of the current study, 8-IsoP levels may

be a marker for oxidative stress in chronic periodontitis.

Further longitudinal prospective studies involving a larger

population are needed to confirm the findings of present study

and to better understand the role of 8-IsoP in the pathogenesis,

and also address the clinical implication and pathological

mechanism of 8-IsoP in periodontal disease progression.

Funding

The present study was partly funded by Colgate research

grant, Colgate Palmolive India limited, Mumbai, India.

Competing interests

The authors report no potential conflict of interests.

Ethical approval

Ethical Approval given by Ethical Committee and Review

Board of Govt Dental College & Research Institute on 23/4/2010

– No. GDCRI/ACM/PG/Ph.D/1/2009–2010.

Acknowledgement

The authors acknowledge Dr B.S. Nanda Kumar, Statistician,

Bangalore, India, for preparing the statistics.

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