A Dağ

A Doğru

F Kaya

F Çağlayan

M Doğru

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Biotechnol. & Biotechnol. Eq. 20/2006/3 169
CREVICULAR FLUID LEVEL OF ELASTASE IN 
TYPE I AND TYPE II DIABETES MELLITUS  
PATIENTS 
 
A. Doğru1, F. Çağlayan2, A. Dağ1, M. Doğru3, F. Kaya1 
Dicle University, Faculty of Dentistry, Department of Periodontology, Diyarbakir, Turkey1 
Hacettepe University, Faculty of Dentistry, Department of Periodontology, Ankara, Turkey2 
Dicle University, Faculty of Science-Art, Department of Chemistry, Diyarbakir, Turkey3 
 
ABSTRACT 
Elastase activity in pocket fluid samples was taken from 120 patients divided into 4 
groups. The groups were identified as Type I DM (Diabetes mellitus) metabolic uncon-
trolled, Type I metabolic controlled, Type II metabolic uncontrolled, and Type II meta-
bolic controlled groups. The samples were evaluated with spectrophotometry. In our study 
we found that, in addition to the clinical data, elastase enzyme activity level and elastase 
enzyme concentration were found to be significantly higher in metabolic uncontrolled dia-
betic groups than in metabolic controlled groups (p<0.05). Furthermore, we found a re-
lationship between total elastase enzyme activity and enzyme concentration in all of the 
four groups. The findings of the study support the relationship between elastase activity 
and the periodontal disease. We may further draw inferences regarding the effects of 
metabolic control levels on the periodontal tissues in diabetics patients. 
 
Introduction 
Diabetes mellitus is a chronic metabolic 
disease that affects 2-10% of general 
population and is characterized by in-
creased blood glucose levels and glucose 
release in urine. Since periodontal disease 
is also common in diabetics, numerous 
studies have investigated the potential link 
between diabetes and periodontal disease 
(1). 
Diabetes mellitus affects more than 100 
million people worldwide and causes 
metabolic complications due to the ab-
sence, shortage or ineffectiveness of the 
insulin hormone combined with hypergly-
cemy. It is classified into two major types: 
juvenile (Type I) and adult (Type II) (1-3). 
Diabetes cause complications throughout 
the body. Oral complications are also 
                                                           
This project is supported by the Dicle University 
Research Project Coordinationship. 
common in people with diabetes. These 
complications that accompany diabetes 
include xerestomia, loss of tooth, gingivitis, 
periodontitis, abces, and cavities. A con-
clusive link between diabetes and perio-
dontal disease has not been established. 
Periodontal diseases are characterized by 
the infections of tissues that support teeth. 
In periodontal disease, tissue breakdown 
occurs in specific periods of time. Identifi-
cation of these periods where the disease is 
active is important as it will help determine 
the current condition of the disease, iden-
tify those at high risk, and correctly deter-
mine the prognosis (4). 
Most of the previous studies on the dia-
betics and periodontal disease based their 
findings on clinical parameters such as 
pocket depth measurement. In recent years, 
however, new methods are also widely be-
ing used including microbiological, immu-
nological, genetic and biochemical me-
thods, in order to determine the active area 
 
Biotechnol. & Biotechnol. Eq. 20/2006/3 170
where tissue breakdown occurs in perio-
dontal disease. Therefore, there is a grow-
ing number of studies that investigate tissue 
breakdown by-products, infectious media-
tors, bacteria and their by-products in total 
saliva, blood, plaque, gingival tissue and 
gingival sulcus fluid (5). 
The contents of GCF (Gingival crevicu-
lar fluid) primarily consist of enzymes, 
cellular elements, electrolytes, organic 
compounds, and antibacterial factors. En-
zymes in GCF may be host or bacterial in 
nature. As a result of many studies on ly-
sosom enzymes, PMN’s (Polimorphonu-
clear leucocytes) have been shown to be 
the main source of a large majority of lytic 
enzymes in GCF. Studies on the develop-
ment of periodontitis show a correlation 
between the active phases of tissue break-
down and PMN’s infiltration towards the 
sulcus area. Higher PMN activity in the 
sulcus area is observed during the break-
down phase of the periodontal disease (6, 
7). Recent studies have indicated that de-
fects in their PMN function causes diabe-
tics to be more prone to bacterial infections 
and that hyperglycemy and weak metabolic 
control increase the risks of the peridontal 
disease, both of which are attributed to the 
defects of PMN function and collagen 
synthesis in diabetes. The presence in GCF 
of indicators for primary granules of neu-
trophils shows the release of enzymes that 
cause protelytic tissue breakdown. One of 
the markers of primary granule release in 
GCF is elastase (8-11). Elastase is a cold 
protease that leads to collagene breakdown 
and that contributes to elastin turn over. It 
has been shown that, during periodontal 
disease, these enzymes are effective in the 
breakdown of building block proteins and 
plasma-based proteins that join the local 
defense reactions (12, 13). 
The goal of this study was to collect 
clinical data that reflect the severity of the 
periodontal disease in Type I and Type II 
diabetics, as well as to determine activity 
levels of elastase enzyme which helps in 
tissue breakdown and to identify, in terms 
of elastase content in GCF, potential diffe-
rences between metabolic controlled and 
metabolic uncontrolled patient groups with 
Type I and Type II DM. 
Materials and Methods 
Study Population 
This study was conducted at Department of 
Endrochronology, Faculty of Medicine at 
Dicle University. A total of 120 patients 
participated in the study, including 62 
women and 58 men who were diagnosed 
with Diabetes mellitus. Patients were ad-
mitted to the study if they were not taking 
medication affecting periodontal status and 
had received no periodontal therapy in the 
preceding 6 months. The classification of 
the subjects with respect to their diabetic 
state was based on the long-term metabolic 
control measured as percentages of glyco-
sylated hemoglobin (HbA1c) level. HbA1c 
values were classified as follows: ≤8.5% 
(good), 8.6-9.9 % (moderate) and ≥ 10.0 % 
(poor) (14). 
Participants were divided into the fol-
lowing four groups according to HbAlc 
levels : 
1. Type I diabetic metabolic uncontrolled 
group: In this group were 30 participants, 
including 16 females and 14 males aged 
12-21. The average age of the group was 
15.2±2.36. 
2. Type I diabetic metabolic controlled 
group: This group consisted of 30 par-
ticipants including 22 females and 8 
males, aged 12-21. The average age of 
the group was 14.9 ±2.59. 
3. Type II diabetic metabolic uncontrolled 
group: This group consisted of 30 par-
ticipants including 12 females and 18 
males, aged 37-67. The average age of 
the group was 52.4±9.46. 
4. Type II diabetic metabolic controlled 
group: This group consisted of 30 par-
ticipants including 12 females and 18 
males, aged 38-67. The average age of 
the group was 50.03±8.99. 
 
Biotechnol. & Biotechnol. Eq. 20/2006/3 171
Determination of Clinical Periodontal 
Status 
During the course of the study, the follow-
ing measurements from the individuals in 
each group were recorded: the Silness-Löe 
plaque index, Löe-Silness gingival index 
(15), and pocket depth measurements .  
Sampling of Gingival Crevicular Fluid 
Pocket fluid samples were taken during the 
morning hours.In order to eliminate the risk 
of contamination with saliva, sites for GCF 
sampling were chosen among maxillary 
teeth in every case. The selected sites was 
isolated with cotton rolls and gently air 
dried. Crevicular fluid was collected onto 
pre-weighed 2Х8 mm filterpaper strips of 
Whatman 3MM chromatography paper 
(Whatman Industries, Dartford, Kent, UK), 
according to the orifice method described 
by Rüdin et al.(16). The paper strips were 
inserted 1mm. into the sulcus/ pocket and 
left in place for 30 seconds. We discarded 
the paper strips that were contaminated 
with blood. The strips were then immedi-
ately weighed with a scientific scale, and 
the minimize the risk of evaporation, paper 
strips placed into Eppendorf tubes were 
firmly wrapped and stored at -20°C. (17). 
Determination of Elastase-like Enzyme 
Activity in GCF samples 
Three hundred (300) μl of saline was added 
to each Eppendorf tube. The tubes were 
vortexed and kept at -4 ºC to maintain the 
extraction of GCF into the saline. Elastase 
activity of GCF was determined using suc-
cinyl-(ala)3 -p-nitroanilide as substrate. The 
activity was measured in 50mM Tris/ HCL 
buffer containing 5 mM CaCl2, 200 mM 
NaCl, and 1 mM substrate. The reaction 
was started by the addition of 0.1 ml of 
GCF extract, and the initial absorbance 
values were measured at 405 nm. The 
mixtures were then incubated 37 ºC for 3 
hours. At the end of the incubation period, 
the absorbance of the assay mixture was 
determined, and absorbance changes were 
calculated using the initial and final rea-
dings. Elastase- like activity of GCF sam-
ples was calculated as the amount of en-
zyme that hydrolyzes 1 nmol of substrate in 
1 minute under the conditions given above. 
Enzyme activity was expressed as enzyme 
concentration and total enzyme activity. 
Enzyme concentration standands for the 
enzyme activity per μl of GCF, and total 
enzyme activity refers to the total activity 
collected onto the paper strips in 30 se-
conds (17). 
Statistical Analysis 
Student test was used in analysis and com-
parison of the groups based on clinical and 
laboratory parameters. Pearson Corelation 
analysis was used in evaluating the rela-
tionships between GCF elastase levels and 
clinical parameters, and between concen-
tration levels and total enzyme activity 
(18). 
Results and Discussion 
Clinical findings: All mouth PD (Probing 
deep), GI (Gingival index) and PI (Plaque 
index) average values in Type I and Type II 
diabetic metabolic uncontrolled groups 
were found to be higher than metabolic 
controlled groups (p<0.05). Results are 
shown in Table 1 and Table 2. 
Laboratory findings: GCF total elastase 
activity and elastase concentration levels 
were found to be significantly higher in 
metabolic uncontrolled patient groups. Af-
ter the groups were compared using the 
one-way variance analysis, differences 
among groups were found to be unimpor-
tant for total elastase activity and elastase 
concentration at Type I and Type II meta-
bolic uncontrolled and Type I and Type II 
metabolic controlled groups (Table 3 and 
Fig. 1). 
Relationships between GCF enzyme le-
vels and clinical data: we used Pearson 
Correlation factor to determine relation-
ships between enzyme activity in gingival 
sulcus fluid and clinical parameters. After 
the analysis, in Type I metabolic uncon-
trolled group, a significant relationship was 
found   between   pocket   depth   and  total  
 
Biotechnol. & Biotechnol. Eq. 20/2006/3 172
TABLE 1  
Averages and standard deviation of clinical and GCF parameters per group 
PD PI GI DOS 
GROUPS 
X SD X SD X SD X SD 
1 2.668 0.256 2.140 0.330 1.956 0.443 0.998 0.313 
2 2.178 0.258 1.804 0.349 1.569 0.432 0.737 0.101 
3 3.674 0.757 2.470 0.469 2.318 0.437 1.075 0.441 
4 2.656 0.452 2.128 0.376 1.857 0.410 0.728 0.111 
 
TABLE 2 
Differences among groups per clinical parameters 
PD PI GI 
GROUPS T p t P t p 
1 & 2 0.490 0.001* 0.335 0.006* 0.387 0.004* 
1 & 3 1.006 0.000* 0.330 0.007* 0.363 0.008* 
1 & 4 0.012 1.000 0.012 0.999 0.099 0.810 
2 & 3 1.496 0.000* 0.665 0.000* 0.750 0.000* 
2 & 4 0.478 0.001* 0.324 0.008* 0.288 0.052 
3 & 4 1.018 0.000* 0.342 0.005* 0.462 0.000* 
 
TABLE 3 
Elastase-like enzyme activity in gingival crevicu-
lar fluid 
GROUPS 
ELA Concentration 
(nmol/min/ml) 
ELA activity 
(U) 
1 195.764 + 64.170 0.269 + 0.300 
2 74.035 + 34.683 0.090 + 0.148 
3 220.430 + 61.257 0.231 + 0.088 
4 96.757 + 57.658 0.093 + 0.137 
 
0
50
100
150
200
250
Group 1 Group 2 Group 3 Group 4  
Fig. 1. Elastase concentration in GCF (nmol/min/ml). 
 
elastase activity (p<0.05). Also in the same 
group, another significant relationship was 
found between GCF volume and total 
elastase enzyme activity level (p<0.05). In 
Type II DM metabolic uncontrolled group, 
a particularly strong relationship was found 
between GCF volume and total elastase 
activity (p<0.001). (Table 4). Total 
elastase like enzyme activity was found to 
be higher metabolic uncontrolled groups 
than metabolic controlled groups (Fig. 2). 
The above-average presence of PMN in 
GCF occurs simultaneously with the loss of 
clinical attachment along the root surface. 
In many studies, it is shown that periodon-
tal pathogens activate PMN’s and that in 
conjunction with oxygen radicals, they start 
the release of granule contents. The pre-
sence of neutrophils indicators in GCF that 
are specific to primary granules is the main 
evidence that enzymes that cause tissue 
breakdown have been released. Elastase is 
one of the indicators of primary granule 
release in GCF (8, 17, 18). 
Our objective in this study was to iden-
tify the clinical parameters and determine 
the GCF elastase enzyme levels that reflect 
the  severity of   the  disease  in  Type I and  
 
Biotechnol. & Biotechnol. Eq. 20/2006/3 173
TABLE 4 
Relationship between GCF enzyme levels and clinical data 
GROUP 1 GROUP 2 GROUP 3 GROUP 4 
 
r p r p r p r p 
PD-ELA activity 0.557 0.039* 0.206 1.000 -0.271 1.000 0.313 1.000 
PD-ELA concentration 0.603 0.012* 0.142 1.000 -0.426 0.531 0.303 1.000 
PI-ELA activity 0.166 1.000 0.273 1.000 -0.331 1.000 0.291 1.000 
PI-ELA concentration 0.082 1.000 0.243 1.000 -0.468 0.257 0.337 1.000 
GI-ELA activity 0.243 1.000 0.383 1.000 -0.102 1.000 0.422 0.562 
GI-ELA concentration 0.278 1.000 0.350 1.000 -0.192 1.000 0.426 0.533 
DOS-ELA activity 0.553 0.043* 0.425 0.538 0.618 0.008* 0.595 0.015* 
DOS-ELA concentration 0.205 1.000 0.400 0.801 -0.299 1.000 0.510 0.111 
 
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Group 1 Group 2 Group 3 Group 4
Elastase-like enzyme activity (Total Activity)
 
Fig. 2. Total ELA (U) observed in GCF. 
 
type II controlled and uncontrolled diabetic 
patients, and their relationship to one ano-
ther. 
There are a number of methods used to 
collect GCF. Paper strips, capillary tubes, 
and gingival washing apparatus can all be 
used to obtain GCF samples. In this study, 
we used the Orifice technique described in 
Rudin et al. whereby paper strips are 
placed at the sulcus entry (16). We first 
grouped the 120 Type I and Type II diabe-
tes patients based on their metabolic con-
trols. We then measured the periodontal 
conditon, pocket depth, plaque index, and 
gingival index of each group to clinically 
establish their peridontal condition. At the 
conclusion, we found the clinical data to be 
higher in metabolic uncontrolled groups 
than in metabolic controlled groups. 
Tervonen and Knuuttila have not found 
statistically significant differences between 
the formations of pockets in diabetics and 
nondiabetics (19). However, they report 
that decreases in the metabolic control in 
diabetics result in increases in the number 
of pockets. A quick survey of the literature 
including Taylor et al. reveals that weak-
ness of the metabolic control in diabetics 
constitutes a risk factor in periodontal di-
seases (20). 
The formulation of the enzymatic content 
of GCF varies in different studies. Total 
enzyme activity (U) refers to the total en-
zyme activity of GCF obtained within a 
well-defined and constant period of time. 
Enzyme concentration is GCF enzyme ac-
tivity per unit volume (17). A 1988 report 
by Lamster et al. found total unit activity of 
gingival pocket fluid enzyme to be a better 
indicator of periodontal disease (21). 
In our study, after comparing elastase en-
zyme to total enzyme activity in Type I and 
Type II diabetics, GCF elastase levels were 
found to be high in metabolic uncontrolled 
groups and the difference was found to be 
statistically significant. Considering the 
fact that metabolic uncontrolled groups 
would have higher clinical index values 
than other groups, total elastase activity 
level can be said to be an indicator of 
periodontal pathology. 
In their 1992 paper, Eley and Cox found 
the total elastase activity to be a major in-
dicator in determining periodontal condi-
tions (22). 
An analysis of our study reveals that the 
 
Biotechnol. & Biotechnol. Eq. 20/2006/3 174
groups with the highest elastase concentra-
tion levels are the metabolic uncontrolled 
groups. In these groups, the highest 
elastase concentrations were found in Type 
II DM metabolic uncontrolled group, with 
Type I DM uncontrolled group showing the 
second highest levels.  
In their studies of elastase concentration 
levels in 11 volunteer patients, Meyle et al. 
determined that the elastase concentration 
levels were affected by oral hygiene (23). 
Similarly in our study, we have found a 
correlation between lower elastase concen-
tration levels and deterioriating periodontal 
conditions in the metabolic control groups. 
The findings of our study sheds light on 
the effects of metabolic control levels on 
periodontal tissues and hints that low 
metabolic control is a potential risk factor 
for the aggressiveness of the disease. Fur-
thermore, our study highlights the need to 
more thoroughly study the enzymatic 
structure of GCF, a resource that is easy to 
obtain and extremely valuable for the 
timely and sufficient treatment of the 
periodontal disease. 
REFERENCES  
1. Fıratlı E., Yılmaz O., Onan U. (1996) J. Clin. 
Periodontol., 23, 362-366.  
2. Iacopino C. (2000) J. Periodontol., 71(8), 375-
384. 
3. Lösche W., Karapetow F., Pohl C., Kocher T. 
(2000) J. Clin. Periodontol., 27, 537-541. 
4. Kawamura M., Fukuda S., Kawabata K., 
Iwamoto Y. (1998) Australian Dental Journal, 43(5), 
315-320.  
5. Smith O.T., Geegan S.J. (1991) J. Clin. Perio-
dontol., 18, 171-176.  
6. Pippin D.J. (1990) Archs. Oral Biol., 35(4), 325-
328. 
7. Pippin D.J., Cobb C.M., Feil P. (1995) J. Perio-
dont. Res., 30, 42-50.  
8. Ataoğlu T., Gürsel M. (1997) Periodontoloji. 
Damla ofset 2. baski.  
9. Carranza F.A., Newman M.G. (1996) Clinical 
Periodontology, 8th Edition, W.B. Saunders Company, 
Philadelphia, London, Toronto, Montreal, Sydney, 
Tokyo, 121-131, 190-192.  
10. Chen H.Y., Cox S.W., Eley B.M., Mantyla P., 
Ronka H., Sorsa T.J. (2000) Clin. Periodontol., 27, 
366-369.  
11. Cimasoni G. (1983) Crevicular Fluid Updated, 
2nd Edit., Basel, Newyork, Krager, 24-26.  
12. Figueredo C.M.S., Gustafsson A. (1998) J. 
Clin. Periodontol., 25, 531-535. 
13. Figueredo C.M.S., Gustafsson A., Asman B., 
Bergström K. (2000) J. Clin. Periodontol., 27, 572-
577. 
14. Tervonen T., Karjalainen K. (1997) J. Clin. 
Periodontol., 24, 505-510. 
15. Löe H., Silness J. (1963) Acta Odontol. Scand., 
21, 533-551. 
16. Rüdin H.J., Overdiek H.F., Rateitschak K.H. 
(1970) Helv. Odont. Acta, 14, 21-26. 
17. Yamalık N., Çağlayan F., Kilinç K., Kilinç A., 
Tümer C. (2000) J. Periodontol., 71, 460-467. 
18. Nergis Y. (1999) Bioistatistik Araştırma İlkeleri. 
D.Ü. Basımevi, Diyarbakır, Turkey.  
19. Tervonen T., Knuuttila M., Pohjamo L., 
Nurkkala H. (1991) Journal of Clinical Periodonto-
logy, 18, 65-68. 
20. Taylor G.W., Burt B.A., Becker M., Genco 
R.J., Shlossman M., Knowler W.C., Petitt D.J. 
(1996) J. Periodontol., 67, 1085-1093.  
21. Lamster I.B., Oshrain R.L., Florello L.A., 
Celenti R.S., Gordon J.M. (1988) J. Clin. Perio-
dontol., 15, 47-52.  
22. Eley B.M., Cox S.W. (1992) J. Periodont. Res., 
27, 62-69. 
23. Meyle J., Zell S., Breck M., Heller W. (1992) J. 
Periodont. Res., 27, 226-231. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CREVICULAR FLUID LEVEL OF ELASTASE IN TYPE I AND TYPE II DIABETES MELLITUS PATIENTS

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CREVICULAR FLUID LEVEL OF ELASTASE IN TYPE I AND TYPE II DIABETES MELLITUS PATIENTS

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